Freezing Characteristics of Cultured Catharanthus roseus (L). G. Don Cells Treated with Dimethylsulfoxide and Sorbitol in Relation to Cryopreservation

The freezing behavior of dimethylsulfoxide (DMSO) and sorbitol solutions and periwinkle (Catharanthus roseus) cells treated with DMSO and sorbitol alone and in combination was examined by nuclear magnetic resonance and differential thermal analysis. Incorporation of DMSO or sorbitol into the liquid growth medium had a significant effect in the temperature range for initiation to completion of ice crystallization. Compared to the control, less water crystallized at temperatures below −30°C in DMSO-treated cells. Similar results were obtained with sorbitol-treated cells, except sorbitol had less effect on the amount of water crystallized at temperatures below −25°C. There was a close association between the per cent unfrozen water at −40°C and per cent cell survival after freezing for 1 hour in liquid nitrogen. It appears that, in periwinkle suspension cultures, the amount of liquid water at −40°C is critical for a successful cryopreservation. The combination of DMSO and sorbitol was the most effective in preventing water from freezing. The results obtained may explain the cryoprotective properties of DMSO and sorbitol and why DMSO and sorbitol in combination are more effective as cryoprotectants than when used alone.     

Cryopreservation of periwinkle,Catharanthus roseus cells cultured in vitro

A procedure for prolonged cryogenic storage of periwinkle cell cultures is described. Cells derived from periwinkle, Catharanthus roseus (L.) G. Don, and subcultured as suspension in 1-B5C nutrient medium have been frozen, stored in liquid nitrogen (–196°C) for 11 weeks, thawed and recultured. Maximal survival was achieved when 3–4 day-old cells precultured for 24 h in nutrient medium with 5% DMSO were frozen at slow cooling rates of 0.5 or 1°C/min prior to storage in liquid nitrogen. The only loss in viability of cells occurred subsequent to treatment with DMSO. Abbreviations: DMSO, dimethylsulfoxide; 2,4-D, 2,4-dichlorophenoxyacetic acid; TTC, triphenyltetrazolium chloride.NRCC No. 20082     

Predict the glass transition temperature of glycerol-water binary cryoprotectant by molecular dynamic simulation

Vitrification is proposed to be the best way for the cryopreservation of organs. The glass transition temperature (T_g) of vitrification solutions is a critical parameter of fundamental importance for cryopreservation by vitrification. The instruments that can detect the thermodynamic, mechanical and dielectric changes of a substance may be used to determine the glass transition temperature. T_g is usually measured by using differential scanning calorimetry (DSC). In this study, the T_g of the glycerol-aqueous solution (60%, wt/%) was determined by isothermal-isobaric molecular dynamic simulation (NPT-MD). The software package Discover in Material Studio with the Polymer Consortium Force Field (PCFF) was used for the simulation. The state parameters of heat capacity at constant pressure (C_p), density (ρ), amorphous cell volume (V_cell) and specific volume (V_specific) and radial distribution function (rdf) were obtained by NPT-MD in the temperature range of 90–270 K. These parameters showed a discontinuity at a specific temperature in the plot of state parameter versus temperature. The temperature at the discontinuity is taken as the simulated T_g value for glycerol–water binary solution. The T_g values determined by simulation method were compared with the values in the literatures. The simulation values of T_g (160.06–167.51 K) agree well with the DSC results (163.60–167.10 K) and the DMA results (159.00 K). We drew the conclusion that molecular dynamic simulation (MDS) is a potential method for investigating the glass transition temperature (T_g) of glycerol–water binary cryoprotectants and may be used for other vitrification solutions.     

A simple and efficient procedure for cryopreservation of embryogenic cells of aromatic Indica rice varieties

Embryogenic suspension cells of two commercially cultivated aromatic Indica rice varieties, Basmati 385 and Pusa Basmati 1, were cryopreserved using a simple one-step freezing procedure that does not require a controlled-rate freezer. The procedure involves osmotic pre-conditioning of cells with mannitol, addition of a cryoprotectant solution consisting of sucrose, dimethyl sulfoxide, glycerol, proline, and modified R₂ medium, cooling to –25°C for 2 h in a freezer, and then storage in liquid nitrogen. After rapid thawing at 45°C, these cultures showed post-thaw cell viability of 5.6 to 10.5% and formed actively dividing, readyto-use cell suspensions in 20–35 d when cultured directly into liquid medium. Plants were regenerated from cell clumps as well as from colonies formed by protoplasts that were isolated from suspension cells re-established from cryopreserved cells, with frequencies higher (54–98%) than, or comparable to, those obtained from three to four-month-old original non-frozen cell cultures. Cell viability and regeneration frequencies of post-thawed Pusa Basmati 1 cultures were similar to those obtained from the suspension cells cryopreserved using the conventional slow-freezing procedure which involves pre-freezing cells to –40°C at the rate of –0.2°C per min prior to immersion in liquid nitrogen. In Basmati 385, however, cells frozen at ––25°C showed lower post-thaw cell viability than those preserved using the slow-freezing procedure, but these cells produced cell suspensions that had greater shoot morphogenetic potential. The study indicates the beneficial effect of this simple freezing procedure, not only for preserving desirable cultured cells but also for an enrichment of embryogenic cells.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - DMSO dimethylsulfoxide - LN liquid nitrogen - MS Murashige and Skoog (1962) medium - NAA -napthaleneacetic acid - pcv packed cell volume - TTC 2,3,5-triphenyltetrazolium chloride     

Cryopreservation of seed of Western Australian native species

The ability of seed of native Western Australian species to be stored using cryopreservation methods was investigated by subjecting seed of 90 native species representing 84 genera and 33 families to storage in liquid nitrogen. Seed of 68 native Western Australian species were germinated after storage in liquid nitrogen for two weeks following treatments which involved direct plunging into liquid nitrogen or slow cooling at 0.4°C min^–1 in 15% dimethyl sulphoxide (DMSO) or slow cooling at 0.4°C min^–1 in 35% DMSO. The largest number of species (37) responded positively to direct plunging without pretreatments, with only 10 species responding to slow cooling in 15% DMSO. Thirty one species had enhanced germination and 10 species depressed germination after any of the liquid nitrogen treatments. There were no trends in a species ability to survive liquid nitrogen storage and freezing regime, moisture content, seed size or taxonomic relatedness. However, hard seeded species belonging to the families Caesalpinaceae and Papilionaceae showed a consistently high degree of tolerance to liquid nitrogen storage. Significant physical damage to seed and cotyledons only occurred in Templetonia retusa (Papilionaecae) and this was alleviated by nicking the seed coat. This study indicates that seed of a large proportion of native Western Australian species may be amenable to storage in liquid nitrogen and that at least 40% of the listed rare and endangered species of Western Australia could be maintained in this way.     

The comparison of sensitivity of motion sickness between retinal degeneration fast mice and normal mice

Recent studies report that a conflict between information from the visual system and vestibular system is one of the main reasons for induction of motion sickness (MS). We may be able to clarify the integration mechanism of visual and vestibular information using an animal model with a visual defect, the retinal degeneration fast (rdf) mouse, and the role of vestibular information in the pathogenesis of MS. The rdf mice and wild-type Kunming mice were subjected to rotary stimulation to induce MS. Conditioned taste anorexia to saccharin solution and behavior score were used to observe the differences in MS sensitivity between two types of mice. The decrease in intake of saccharin solution and the behavior score in rdf mice were greater than those in normal mice. After rotatory stimulation, the reduction of intake mass and the behavior score were greater in rdf mice compared to those of normal mice. The rdf mice were more sensitive to rotation than normal mice. We conclude that visual information plays a role in the pathogenesis of MS. Visual information and vestibular information impact each other and integrate through certain channels in the central nervous system in mice.     

Differentiation of the multiple S- and N-oxide-reducing activities ofEscherichia coli

InEscherichia coli, several terminal reductases catalyze the reduction of S- and N-oxide compounds. We have used mutants missing either the constitutive dimethylsulfoxide (DMSO) reductase,dmsABC, and/or the inducible trimethylamine N-oxide (TMAO) reductase,torA, to define the roles of each reductase. These studies indicated that the constitutive DMSO reductase can sustain growth on DMSO, TMAO, methionine sulfoxide (MetSO), and other N-oxide compounds. Only one inducible TMAO reductase is expressed inE. coli, and this enzyme sustains growth on TMAO but not DMSO or MetSO. Characterization of atorA ^–, dms^–double mutant revealed that adenosine N-oxide (ANO) reductase is specifically required for anaerobic respiration on ANO in this mutant.     

Cryopreservation of wheat suspension culture and regenerable callus

Wheat (Triticum aestivum L. cv. Norstar) suspension cultures and regenerable calli initiated from immature embryos can be cryopreserved in liquid nitrogen temperature (–196°C) by slow freezing (0.5°C/min) in the presence of a mixture of DMSO and sucrose or sorbitol. Cold hardening or ABA treatment before cryopreservation increased the freezing resistance and improved the survival of wheat suspension culture in liquid nitrogen. Callus culture, established from immature embryos, prefrozen in 5% DMSO and 0.5M sorbitol survived liquid nitrogen storage and resumed plant regeneration after thawing. The results confirm the feasibility of long term preservation of wheat embryo callus by cryopreservation and retention of plant regeneration ability.Abbreviations ABA Abscisic acid - 2,4-D 2,4-Dichlorophenoxyacetic acid - DMSO Dimethylsulfoxide - LN Liquid nitrogen - TTC 2,3,5-triphenyltetrazolium chloride NRCC No. 23850.     

A new one-step method for the cytochemical localization of ouabain-sensitive,potassium-dependent p-nitrophenylphosphatase activity

Summary A new one-step method for the light and electron microscopic localization of the ouabain-sensitive, K-dependent p-nitrophenylphosphatase (K-NPPase) activity of the Na-K-ATPase complex is introduced. The incubation medium contains p-nitrophenylphosphate (NPP) as substrate, lead citrate as the capture reagent, and dimethylsulfoxide (DMSO) as an activator. It is usable at the optimal pH of the K-NPPase, which is about pH 9.0 in the presence of 25% DMSO. The effects of fixation, lead concentration, and DMSO on the enzyme activity were studied using rat kidney as a test tissue. The fixation of tissues in a mixture of 2% paraformaldehyde and 0.5% glutaraldehyde for 60 min at 0°–4° C preserved 45% of the enzyme activity. In the absence of DMSO, lead citrate (4.0 mM) caused 82% inhibition of the enzyme activity in fixed tissue. However, the addition of DMSO (25%) caused about 3-fold activation of the remaining activity. Cytochemical demonstration of the ouabain-sensitive K-NPPase activity was successfully made by this method at both light and electron microscopic levels.This study was supported partly by grants-in-aid for scientific reasearch from the Ministry of Education, the Japanese Government (Nos. 244016 and 337001)Part of this paper was presented at the 19th Annual Meeting of the Japan Society of Histochemistry and Cytochemistry held in Gifu, November 1–2, 1978 (Mayahara et al. 1979a), and the 35th Annual Meeting of the Japanese Society of Electron Microscopy, held at Takarazuka, May 23–25, 1979 (Mayahara et al. 1979b)     

Morphological differentiation of neuroblastoma cells induced by dimethylsulfoxide

Morphological features of neuroblastoma cells grown in culture in the presence of dimethylsulfoxode (DMSO) were studied. Morphological differentiation, expressed as the appearance of long axon-like processes (neurites), an increase in size of the cells, and inhibition of cell division, was observed in neuroblastoma cells of line C 1300, subline N-18-TG₂A₁, incubated in medium containing 1% DMSO. In the early stages of culture in normal growth medium the cells possess primary features of morphological differentiation. Quantitative criteria for the development of these features depending on duration of culture in modified medium were worked out. An increase in the total length of the neurites of cells differentiating under the influence of DMSO is a linear function of time. The rate of growth of the neurites is 20.0±3.0 µ/h. The area of cross-section of the soma of the differentiated cells is 6–7 times greater than the corresponding parameter in the control. An increase in the DMSO concentration in the culture medium (1.5 and 2.0%) does not induce rapid growth of the neurites or an increase in size of the cell soma, but it does block mitosis. Characteristics of morphological differentiation of neuroblastoma cells are compared with probable functional changes in these cells.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 16, No. 4, pp. 519–527, July–August, 1984.     

Exploring the antioxidant potential of lignin isolated from black liquor of oil palm waste

This study was conducted to evaluate the potential antioxidant activity of lignin obtained from black liquor, a hazardous waste product generated during the extraction of palm oil. Antioxidant potential of the extracted lignin was evaluated by dissolving the extracted samples in 2 different solvent systems, namely, 2-methoxy ethanol and DMSO. Results revealed high percent inhibition of the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical in the lignin sample dissolved in 2-methoxy ethanol over DMSO (concentration range of 1–100 μg/ml). Lignin extracted in 2-methoxy ethanol exhibited higher inhibition percentage (at 50 μg/ml, 84.2%), whereas a concentration of 100 μg/ml was found to be effective in the case of the DMSO solvent (69.8%). Fourier transform infrared (FTIR) spectrometry revealed that the functional groups from the extracted lignin and commercial lignin were highly similar, indicating the purity of the lignin extracted from black liquor. These results provide a strong basis for further applications of lignin in the food industry and also illustrate an eco-friendly approach to utilize oil palm black liquor. To cite this article: R. Bhat et al., C. R. Biologies 332 (2009).     

The vertebrate pineal hormone melatonin is produced by the brown alga Pterygophora californica and mimics dark effects on growth rate in the light

Melatonin, a methoxylated indoleamine, plays a role as a mediator of darkness in animals as well as in the unicellular alga Gonyaulax polyedra Stein and was recently detected in higher plants. We report on the first finding of melatonin in a multicellular alga, the brown alga Pterygophora californica Rupr. Melatonin was identified in juvenile sporophytes of P. californica by two independent methods, reverse-phase high-performance liquid chromatography (HPLC) with electrochemical detection, and radioimmunoassay. Another indolic metabolite, 5-methoxytryptophol, was also indentified by HPLC. The rapid decline of growth rate upon the onset of darkness in P. californica is mimicked by melatonin in the light, with increasing efficiency from 5 × 10^–5M to 5 × 10^–4M, while no effect was obtained at 10^–5M.Abbreviations ANOVA analysis of variance - DMSO dimethyl sulfoxide - LD light-dark cycle K.L. and A.W. thank Petra Kadel for help with algal cultivation and evaluation of the experiments.     

Extraction and quantitation of astaxanthin from Phaffia rhodozyma

Summary The rapid, quantitative release of astaxanthin and other carotenoids from the yeast Phaffia rhodozyma is described. Hashed cells are ruptured with dimethylsulfoxide (DMSO) and carotenoids extracted into an organic solvent. Extraction and spectrophotometric quantitation of total carotenoids is rapid, reproducible and only small volumes (0.1–2 ml) of culture are required. HPLC analysis in normal phase silica gel column indicates that astaxanthin comprises 65–95% of the total pigmented carotenoids of P. rhodozyma.     

Emulsan quantitation by Nile red quenching fluorescence assay

A Nile red fluorescent technique to quantify 20–200 g ml^–1 of emulsan was developed. Nile red dissolved in DMSO showed an adsorption peak at 552 nm, and emission peak at 636 nm, with molar extinction coefficient of 19,600 cm^–1 M^–1. Nile red fluorescence in DMSO was proportionally quenched by emulsan and the quenching was time-dependent. The assay was used to follow the production of emulsan by cultures of Acinetobacter venetianus RAG-1.     

Somatic embryogenesis of the banana hybrid cultivar FHIA-18 (AAAB) in liquid medium and scaled-up in a bioreactor

The development of somatic embryos in liquid culture medium has a number of advantages for large-scale propagation of plants. This paper describes an improved system for the mass propagation via somatic embryogenesis of the banana hybrid cultivar FHIA-18 (AAAB). Explants from immature male flowers were used to form high frequency embryogenic tissue, this tissue was then used to establish embryogenic cell suspensions in a basic MS medium plus 1.0 mg l^–1 biotin, 100 mg l^–1 glutamine, 100 mg l^–1 malt extract (Sigma), 1.0 mg l^–1 2,4-D and 45 g l^–1 sucrose. Secondary multiplication of somatic embryos was achieved in liquid media in rotary shaker and in bioreactors. The number of embryos per litre obtained with 80.0% DO₂ and effects of pH were also studied. A high regeneration percentage of plants were obtained (89.3%) in only 1 month of culture, somatic embryos were then placed to germinate in temporary immersion systems and field testing of somaclonal variation.     

Development of a microporous membrane liquid–liquid extractor for organophosphate esters in human blood plasma: identification of triphenyl phosphate and octyl diphenyl phosphate in donor plasma

An extractor has been developed for microporous membrane liquid–liquid extraction (MMLLE) of lipophilic xenobiotics at trace levels in biological fluids. This new construction allows the sample phase to be stirred, while the organic phase is pumped. The extractor was evaluated using human blood plasma with added organophosphate esters. The size exclusion properties of the membrane reduced lipid co-extraction by 94% compared to ordinary liquid–liquid extraction. In combination with a solid-phase extraction (SPE) step, the method was shown to remove plasma lipids efficiently and thus allow gas chromatographic separation of the compounds. The clean-up method described, including the SPE step, showed a high level of reproducibility, and recoveries of between 72 and 83% were obtained for five of the organophosphate esters after a 200-min extraction period. Using this technique, triphenyl phosphate and an isomer of octyl diphenyl phosphate were detected in human plasma obtained from blood donors. The concentration of triphenyl phosphate ranged between 0.13 and 0.15 μg/g plasma.     

Separation and identification of corticosterone metabolites by liquid chromatography–electrospray ionization mass spectrometry

High-performance liquid chromatography coupled to atmospheric pressure ionization–electrospray ionization mass spectrometry (API–ESI–MS) was investigated for the analysis of corticosterone metabolites; their characterization was obtained by combining the separation on Zorbax Eclipse XDB C₁₈ column (eluted with a methanol–water–acetic acid gradient) with identification using positive ion mode API–ESI–MS and selected ion analysis. The applicability of this method was verified by monitoring the activity of steroid converting enzymes (20β-hydroxysteroid dehydrogenase and 11β-hydroxysteroid dehydrogenase) in avian intestines.     

Dimethyl sulfoxide-induced high enantioselectivity of subtilisin Carlsberg for hydrolysis of ethyl 2-(4-substituted phenoxy)propionates

The enantioselectivity for subtilisin-catalyzed hydrolysis of ethyl 2-(4-substituted phenoxy)propionates in an aqueous buffer solution was improved by addition of DMSO (54–56% v/v). On the basis of the conformational change of subtilisin Carlsberg observed for FT-IR and CD spectra, the high enantioselectivity for subtilisin-catalyzed hydrolysis of racemic ethyl 2-(4-ethylphenoxy)propionate could be related to a partial decrease of the tertiary structure of the enzyme protein arising from an increase of the ratio of DMSO in the reaction medium. This mechanistic model for the enantiorecognition can also be supported by the discussion based on the value of the Michaelis constant (K _m) obtained for each enantiomer of the substrate.     

ULTRATHIN FROZEN SECTIONS : I. Methods and Ultrastructural Preservation

A relatively simple method for obtaining ultrathin, frozen sections for electron microscopy has been developed. Tissues, cultured cells, and bacteria may be employed. They are fixed in 1.25–4% glutaraldehyde for 1–4 hr, are washed overnight in buffer at 3°C, and are embedded in 20% thiolated gelatin or pure gelatin. Before sectioning they are partially dehydrated in 50% glycerol, frozen in liquid nitrogen on a modified tissue holder, and subsequently maintained at -70°C with dry ice. Finally, they are sectioned very rapidly with glass knives on a slightly modified Porter-Blum MT-1 microtome in a commercial deep-freeze maintained at -35°C and are floated in the trough of the knife on a 40% solution of dimethylsulfoxide (DMSO). The sections are picked up in plastic loops and transferred to distilled water at room temperature for thawing and removal of the DMSO, placed on grids coated with Formvar and carbon, air-dried, and stained with phosphotungstic acid, sodium silicotungstate, or a triple stain of osmium tetroxide, uranyl acetate, and lead. Large flat sections are obtained in which ultrastructural preservation is good. They are particularly useful for cytochemical studies.     

Cryopreservation of sugarbeet germplasm

Meristems aseptically isolated from shoots developed on sugarbeet (Beta vulgaris L.) inflorescences were precultured on modified MS agar medium containing 19.4 M 6-benzylaminopurine, 6 M triiodobenzoic acid, and supplemented with 5% DMSO. After two days the meristems were transferred to liquid modified MS medium and the cryoprotectants sorbitol and DMSO added in varying concentrations. The meristems were frozen to –40°C and stored in liquid nitrogen. Growth resumed when the meristems were quick-thawed at 39°C.