Rapid Immersion Research Articles

Metabolic comparison of cryopreserved and normal cells from Tabernaemontana divaricata suspension cultures

A cryopreservation protocol for Tabernaemontana divaricata suspension cell cultures (6 Div BW 101) was established. Cells were precultured in MS medium supplemented with 0.5 and 0.33 M mannitol for 2 or 3 days following with incubation in MS media with a mixture of 1 M sucrose, 0.5 M glycerol, 0.5 M DMSO, and 0.04 M L-proline as cryoprotectant in an ice bath for 20 min. The cells were transferred into 2 ml cryogenic vials and then, the vials were put into the cryogenic container prior to placing at a −80 °C freezer for 4 h followed by rapid immersion in liquid nitrogen. The cells were transferred without washing a MS medium solidified with 7% (w/v) agarose. Cells that were precultured 3 days after subculturing in MS medium supplemented with 0.5 M mannitol for 3 days, showed growth recovery. Metabolic profiling of control and cryopreserved Tabernaemontana divaricata cells was performed by 1H-NMR spectroscopy combined with PCA, GC, and HPLC. Differences of metabolic accumulation were found in the level of several amino acids, carbohydrates, and fumaric acid. However, the levels of the main alkaloid precursor tryptamine did not change.

Deep-sea caging of the mussel Mytilus galloprovincialis: Potential application in ecotoxicological studies

We experimented with caging the Mediterranean mussel (Mytilus galloprovincialis) at various depths for 69 d to measure basic physiological parameters, histological response and bio-accumulation of contaminants in a deep-sea contaminated area. In preliminary experiments, we demonstrated, under artificial pressure conditions, the ability of mussels Mytilus galloprovincialis to tolerate rapid immersion (at a speed of up to 120 m min−1). In situ experiments were performed using submerged lines enabling mussels to be maintained at depths ranging of 40–1550 m with survival rates ranging from 80 to 38%, respectively. No significant differences in condition indexes were observed between treated and control specimens. However, histological observations demonstrated a clear reduction in thickness of the digestive epithelium with increasing depth exposure. By determining the contaminants in caged mussels, we found the following values for chromium accumulation: 27.4 μg g−1 dry weight at 580 m depth and 9.8 μg g−1 dry weight at 1550 m. Selected stations were located downstream of an industrial effluent at 420 m. The biological and environmental consequences of deep-sea contamination demonstrate the suitability of caged mussels for monitoring contaminant accumulation.

Cryopreservation of chestnut by vitrification of in vitro-grown shoot tips

Plants of European chestnut (Castanea sativa) have been consistently recovered from cryopreserved in vitro-grown shoot apices by using the vitrification procedure. Factors found to influence the success of cryopreservation include the source of the shoot tips (terminal buds or axillary buds), their size, the duration of exposure to the cryoprotectant solution, and the composition of the post-cryostorage recovery medium. The most efficient protocol for shoot regrowth employed 0.5–1.0 mm shoot tips isolated from 1 cm-long terminal buds that had been excised from 3–5-wk shoot cultures and cold hardened at 4°C for 2 wk. The isolated shoot tips were precultured for 2d at 4°C on solidified Gresshoff and Doy medium (GD) supplemented with 0.2M sucrose, and were then treated for 20 min at room temperature with a loading solution (2M glycerol+0.4M sucrose) and for 120 min at 0°C with a modified PVS2 solution before rapid immersion in liquid nitrogen (LN). After 1 d in LN, rapid rewarming and unloading in 1.2M sucrose solution for 20 min, the shoot tips were plated on recovery medium consisting of GD supplemented with 2.2 μM benzyladenine, 2.9 μM 3-indoleacetic acid, and 0.9 μM zeatin. This protocol achieved 38–54% shoot recovery rates among five chestnut clones (three of juvenile origin and two of mature origin), and in all cases plant regeneration was also obtained.

A Novel Mode of Gleevec Binding Is Revealed by the Structure of Spleen Tyrosine Kinase

Spleen tyrosine kinase (Syk) is a non-receptor tyrosine kinase required for signaling from immunoreceptors in various hematopoietic cells. Phosphorylation of two tyrosine residues in the activation loop of the Syk kinase catalytic domain is necessary for signaling, a phenomenon typical of tyrosine kinase family members. Syk in vitro enzyme activity, however, does not depend on phosphorylation (activation loop tyrosine --> phenylalanine mutants retain catalytic activity). We have determined the x-ray structure of the unphosphorylated form of the kinase catalytic domain of Syk. The enzyme adopts a conformation of the activation loop typically seen only in activated, phosphorylated tyrosine kinases, explaining why Syk does not require phosphorylation for activation. We also demonstrate that Gleevec (STI-571, Imatinib) inhibits the isolated kinase domains of both unphosphorylated Syk and phosphorylated Abl with comparable potency. Gleevec binds Syk in a novel, compact cis-conformation that differs dramatically from the binding mode observed with unphosphorylated Abl, the more Gleevec-sensitive form of Abl. This finding suggests the existence of two distinct Gleevec binding modes: an extended, trans-conformation characteristic of tight binding to the inactive conformation of a protein kinase and a second compact, cis-conformation characteristic of weaker binding to the active conformation. Finally, the Syk-bound cis-conformation of Gleevec bears a striking resemblance to the rigid structure of the nonspecific, natural product kinase inhibitor staurosporine.

Cryopreservation by encapsulation-dehydration of ‘Christmas bush’ (Ceratopetalum gummiferum) shoot tips

Abstract Christmas bush (Ceratopetalum gummiferum Sm) is a shrubby tree species of the east coast of New South Wales in Australia. It is much prized as a cut flower crop because of its bright, pinky red floral calyces. New varieties are being developed, the storage of which is an important issue. In this study, it was shown that shoot tips sampled from in vitro plantlets withstood cryopreservation using the encapsulation–dehydration technique. The protocol leading to optimal regrowth was the following: excised shoot tips were pretreated for 1 d in the dark on hormone-free Murashige and Skoog (MS) medium with 0.3 M sucrose, then encapsulated in 3% calcium alginate and precultured in liquid MS medium with 0.5 M sucrose for 3 d. Precultured beads were dehydrated for 6 h in the air current of the laminar flow cabinet to 24.3% moisture content (fresh weight basis) before rapid immersion in liquid nitrogen. Under these conditions, regrowth of shoot tips after cryopreservation reached 61.4%. Regrowth of cryopres...

PROPAGATION OF PLANTS IN BIOREACTORS: PROSPECTS AND LIMITATIONS

Bioreactors provide a rapid and efficient plant micropropagation system but as yet are not fully exploited commercially for agricultural crops and forest trees. The culture system was applied to herbaceous and woody plant species utilizing liquid media to overcome intensive manual handling. Large-scale liquid cultures were used for propagation through organogenesis or somatic embryogenesis pathways depending on the species. Bioreactors have several advantages over agar cultures with a better control of the culture conditions. Direct contact of the plant tissue with the medium, optimal nutrient and growth regulators supply, continuous aeration and circulation, filtration of the medium for controlling exudates and contamination and the production of clusters of buds, meristems or protocorms for automated dispensing. The major issue imposed by liquid media in bioreactors is the phenomenon of hyperhydricity, morphogenic shoot and leaf malformation, due to the continuous immersion of the tissue in the medium. The malformations are manifested in glossy hyperhydrous leaves with distorted anatomy. The very nature of liquid tissue culture is an imposing stress to which the plants respond to the environmental signals in developmental aberration. The submerged tissue was found to exhibit oxidative stress symptoms, with elevated levels of reactive oxygen species that were associated with a change in anti-oxidant enzyme activity. These changes greatly affected the anatomy and physiology of the plants and their survival after transplanting. Two major solutions for malformation control were proposed: the use of growth retardants to control rapid proliferation and temporary immersion bioreactors. Growth retardants reduced water uptake during cell proliferation, decreased vacuolation and rapid growth, shortened stems and inhibited leaf expansion, inducing the formation of clusters. In tuber, bulb and corm producing plants growth retardants were found to enhance storage organs formation. Temporary immersion bioreactors were used to provide an improved aeration system to reduce hyperhydricity, by shortening plant duration in the liquid phase. The frequency of immersion and the medium composition improved plant development. Future prospects of micropropagation in bioreactors for optimal plant production will depend on both basic and applied studies. The former contributing to our understanding of plant responses to microenvironment signals to overcome the limitations of liquid cultures and the latter to provide specific culture manipulation to control the morphogenesis of plants in vitro improving survival ex vitro.

Comparison of efficacy of different cryoprotectant solutions for mouse ovarian tissue

Comparison of efficacy of different cryoprotectant solutions for mouse ovarian tissue

Structural Analysis of the Protein/Lipid Complexes Associated with Pore Formation by the Bacterial Toxin Pneumolysin

Pneumolysin, a major virulence factor of the human pathogen Streptococcus pneumoniae, is a soluble protein that disrupts cholesterol-containing membranes of cells by forming ring-shaped oligomers. Magic angle spinning and wideline static (31)P NMR have been used in combination with freeze-fracture electron microscopy to investigate the effect of pneumolysin on fully hydrated model membranes containing cholesterol and phosphatidylcholine and dicetyl phosphate (10:10:1 molar ratio). NMR spectra show that the interaction of pneumolysin with cholesterol-containing liposomes results in the formation of a nonbilayer phospholipid phase and vesicle aggregation. The amount of the nonbilayer phase increases with increasing protein concentration. Freeze-fracture electron microscopy indicates the coexistence of aggregated vesicles and free ring-shaped structures in the presence of pneumolysin. On the basis of their size and analysis of the NMR spectra it is concluded that the rings are pneumolysin oligomers (containing 30-50 monomers) complexed with lipid (each with 840-1400 lipids). The lifetime of the phospholipid in either bilayer-associated complexes or free pneumolysin-lipid complexes is > 15 ms. It is further concluded that the effect of pneumolysin on lipid membranes is a complex combination of pore formation within the bilayer, extraction of lipid into free oligomeric complexes, aggregation and fusion of liposomes, and the destabilization of membranes leading to formation of small vesicles.

Contractile activation characteristics of single permeabilized fibres from levator palpebrae superioris, orbicularis oculi and vastus lateralis muscles from humans.

1. We investigated the contractile activation characteristics of single membrane-permeabilized fibres from the following muscles from humans: the levator palpebrae superioris (LPS), an extraocular muscle; the orbicularis oculi (OO), a facial muscle; and the vastus lateralis (VL), a major muscle of the thigh. 2. Single permeabilized muscle fibres were isolated from each of the different muscles, attached to a sensitive force transducer and activated by rapid immersion in buffered solutions of varying [Ca2+] and [Sr2+]. Fibres were allocated into discrete populations based on their contractile characteristics, including their differential force responses during Ca2+ and Sr2+ activation. 3. With the exception of one fibre from the LPS, all 152 fibres sampled from the three different human muscles could be classified into either population I (slow, type I) or population II (fast, type II) based on their force-pCa(pSr) relations. The LPS muscle fibre which was unable to be classified into the two major fibre populations displayed a combination of the typical force-pCa(pSr) relations for mammalian fast and slow muscle fibres. 4. Although fibres from the LPS, OO and VL muscles had similar differential sensitivities to Ca2+and Sr2+, the steepness of the force-pCa(pSr) curves for fibres from the LPS and OO muscles were highly variable compared with those for fibres from the VL muscle. Specific forces (N cm-2) of the smaller diameter fibres from the LPS and OO muscles were significantly lower than those of fibres from the VL muscle. 5. The differences in the contractile activation characteristics between fibres from the VL muscle and those of fibres from facial (OO) muscles and extraocular (LPS) muscles, reflect the differences in their fibre composition that are responsible for their functional specificity.

Near-drowning.

Near-drowning.

The influence of the soil conditioner `Agri-SC' on splash detachment and aggregate stability

The influence of the soil conditioner `Agri-SC' on splash detachment and water-stable aggregation of an erodible clay Vertisol from Oahu, HI, was assessed. Laboratory rainfall simulation was used to assess splash detachment from soil treated with 0 (untreated control), 0.3, 3.0, 30, and 300 l ha −1 of Agri-SC. Results indicated that the quantity of sediment splashed was significantly lower for Agri-SC application rates of 0.3 and 3.0 l ha −1 (rates are equivalent to 1 and 10 times the manufacturer's recommended rates, respectively), than for the control, or for Agri-SC applied at 30 and 300 l ha −1 (100 and 1000×, respectively). A second experiment was designed to test the influence of Agri-SC on water-stable aggregation of the Vertisol. Aggregates were subjected to rapid immersion in solution, shaken and washed through a series of sieves. Data indicated that there were no statistically significant differences in geometric mean aggregate diameter between the untreated and treated aggregates. The effect of the active ingredient, ammonium laureth sulfate (an anionic surface active agent) on splash and erodibility are discussed. These preliminary results indicate that further testing of Agri-SC is warranted on a variety of soils with different textures and mineralogies.

Structure of the Erythrocyte Membrane Skeleton as Observed by Atomic Force Microscopy

The structure of the membrane skeleton on the cytoplasmic surface of the erythrocyte plasma membrane was observed in dried human erythrocyte ghosts by atomic force microscopy (AFM), taking advantage of its high sensitivity to small height variations in surfaces. The majority of the membrane skeleton can be imaged, even on the extracellular surface of the membrane. Various fixation and drying methods were examined for preparation of ghost membrane samples for AFM observation, and it was found that freeze-drying (freezing by rapid immersion in a cryogen) of unfixed specimens was a fast and simple way to obtain consistently good results for observation without removing the membrane or extending the membrane skeleton. Observation of the membrane skeleton at the external surface of the cell was possible mainly because the bilayer portion of the membrane sank into the cell during the drying process. The average mesh size of the spectrin network observed at the extracellular and cytoplasmic surfaces of the plasma membrane was 4800 and 3000nm2, respectively, which indicates that spectrin forms a three-dimensionally folded meshwork, and that 80% of spectrin can be observed at the extracellular surface of the plasma membrane.

A new approach to determining water stable aggregation

A new method is introduced to measure water stability of soil aggregates. The wrist‐action shaker is a simple, inexpensive tool that provides highly accurate data for the assessment of soil erodibility. Three soils from Hawaii (two Oxisols and one Vertisol) with different mineralogies, management histories, and potassium (K)‐factors were examined in this study. Six indices of water stable aggregation were determined after rapid immersion of air‐dry aggregates, followed by gentle wet‐sieving. Single‐sieve indices of percent water stable aggregates (WSA) < 0.063 mm, > 0.25 mm, and > 1.00 mm, were highly correlated. Additionally, these indices were highly correlated with three multiple sieve indices, namely geometric mean aggregate diameter (GMAD), arithmetic mean aggregate mass diameter (MAMD), and the coarse‐to‐fine index (CFI = % WSA > 1.00 mm / % WSA < 0.063 mm). Analysis of WSA data indicated that the relative soil erodibility ranking, from high to low, would be: Lualualei Vertisol > Molokai Oxisol > Kaneloa Oxisol. Discriminant analysis using GMAD and % WSA > 1.00 mm correctly classified 55 of 56 soil samples into their respective soil series.

Novel Microwave Technology for Cryopreservation of Biomaterials by Suppression of Apparent Ice Formation

Ice formation inside or outside cells has been proposed to be a factor causing cryoinjury to cells/tissues during cryopreservation. How to control, reduce, or eliminate the ice formation has been an important research topic in fundamental cryobiology. The objective of this study was to test a hypothesis that the coupled interaction of microwave radiation and cryoprotectant concentration could significantly influence ice formation and enhance potential vitrification in cryopreservation media at a relative slow cooling rate. Test samples consisted of a series of solutions with ethylene glycol (a cryoprotectant) concentration ranging from 3 to 5.5M.A specific microwave resonant cavity was built and utilized to provide an intense oscillating electric field. Solutions were simultaneously exposed to this electric field and cooled to −196°C by rapid immersion in liquid nitrogen. Control samples were similarly submerged in liquid nitrogen but without the microwave field. The amount of ice formation was determined by analysis of digital images of the samples. The morphology of the solidified samples was observed by cryomicroscopy. It was found that ice formation was greatly influenced by microwave irradiation. For example, ice formation could be reduced by roughly 56% in 3.5Methylene glycol solutions. An average reduction of 66% was observed in 4.5Msolutions. Statistical analysis indicated that the main effects of microwave and ethylene glycol concentration as well as the interaction between these two factors significantly (P< 0.01) influenced ice formation amount, confirming the hypothesis. This preliminary study suggests that a combined use of microwave irradiation and cryoprotectant might be a potential approach to control ice formation in cells/tissues during the cooling process and to enhance vitrification of these biomaterials for long-term cryopreservation.

Cryopreservation of Somatic Embryos: A Tool for Germplasm Storage and Commercial Delivery of Selected Plants

The development of long-term preservation methods of somatic embryos was investigated as a contribution to synthetic seed technology, Thus, cryopreservation in liquid nitrogen was attempted with somatic embryos of two species. Coffea canephora Pierre and Daucus carota L. Cryopreservation of carrot embryos simply required a 21-h sucrose pretreatment followed by a prefreezing step at -20°C. A freeze-hardening treatment consisting of embryo culture in the presence of high sucrose molarity and 1 μM ABA was developed for coffee embryos. Following desiccation at 75% relative humidity (RH) and +24°C for 7 d, embryos were frozen by rapid immersion in liquid nitrogen. Direct regrowth of frozen-thawed embryos was obtained for the majority of both carrot and coffee embryos. Even large carrot embryos were able to survive cryopreservation. The size of embryos and the age of the embryogenic strain from which the embryos originated determined their ability to develop into normal plantlets. Direct regrowth without any secondary callogenesis and/or embryogenesis is a prerequisite for practical use of preserved embryos either in a nursery or through direct planting.

Improvement of the simple and rapid immersion method of soil water determination

Rapid and accurate determinations of soil water content are important in many agricultural and hydrologic situations. In this article, an improved method is proposed, which allows a quick and acurate determination of soil water content under both laboratory and field conditions without resorting to sophisticated equipment. A 500 cm 3 erlenmeyer with an adapted level marker was employed for rapid determination of soil water content. The principle involved is to weigh the flask when filled with moist soil and completed to level with water. An operation, which involves a single calibration of the flask with the same, but dry soil, eliminates the need of particle density determination. For several soils, results obtained with the new methodology deviated less than 1% from results of the traditional oven-drying method.

X-ray microanalysis of intracellular ions in epithelial tissues

Electron probe x-ray microanalysis provides a powerful tool in studying transepithelial ion transport mechanisms as it allows the quantitative detection of diffusible ions on a cellular and subcellular scale. The high spatial resolution afforded by this method is of particular advantage in epithelial tissues that are composed of different cell types and cases in which the transcellular transport pathway might involve different intracellular compartments. In the following, the methods applied in several studies are described, with emphasis on some recent modifications.The tissues are frozen by rapid immersion in liquified ethane kept at -180°C. Support systems for different epithelial preparations have been developed to facilitate freezing and maintain the functional state of the epithelium immediately up to the moment of freezing. A fluid-clamp technique is used to mount the tissue for sectioning (Figure 1). Sections are cut dry at -140°C and a nominal thickness of 200 nm using 40° glass knifes. The sections are picked up from the back of the knife by a fire-polished wire probe (1 μm tip diameter) and placed on a parlodion film which is suspended over a 2 mm opening in a special beryllium carrier disc.

A Simple Procedure for the Cryopreservation of Hydrated Embryonic Axes of Pisum sativum

Pea seeds were imbibed for 4 hours or set to germinate for 72 hours, after which the axes were excised. A protocol for rapid freezing of this material was established by determining the most suitable pre-treatments that might effect vitrification of the samples. The procedures included redehydration and variation in exposure time to different combinations of glycerol with other cryoprotectants (DMSO, sucrose, sorbitol and proline). Irrespective of the imbibition time, successful retrieval from liquid nitrogen (47.5% of 4-hour-imbibed and 25% of 72-hour-imbibed axes) was obtained by use of a cryoprotectant miXture of glycerol and sucrose, followed by a halving of the moisture content, rapid immersion in liquid nitrogen, and rapid thawing in warm liquid medium followed by plating out onto solid growth medium. Post-freezing treatments such as removal of cryoprotectants by washing or addition of proline and! or activated charcoal to the growth medium did not improve the recovery of the 4-hour-imbibed material. The results indicate that using simple, rapid and inexpensive pre-treatments, relatively large and partially hydrated plant material has the potential to be conserved in liquid nitrogen.

Localization of the cystic fibrosis gene product in a transfected Sf9 insect cell line

The cystic fibrosis gene product (CFTR) is thought to be either directly or indirectly involved with Cl- conductance in epithelial cells in which it is produced. Utilizing the Baculovirus expression vector system, CFTR has been expressed in Sf9 insect cells which in turn display a regulated Cl- conductance employing the same techniques as those used to demonstrate Cl- conductance in epithelial cells. This was not observed in mock-infected Bgalactosidase producing or non-infected Sf9 cells.Two cell lines, one infected with CFTR expressing recombinant baculovirus and the other infected with B-galactosidase expressing recombinant baculovirus were fixed in 2% paraformaldehyde and 0.001% glutaraldehyde in 0.1 M HEPES buffer, PH7.2 for 2 hours. They were then rinsed several times with buffer, infiltrated with 2% agarose and infused with 2.3 M sucrose for at least 24 hours. The samples were then frozen by rapid immersion in liquid nitrogen cooled FREON 22 and ultrathin cryosections cut with a cryoultramicrotome and immunogold labelled with purified monclonal antibodies against CFTR (1) using the method described by Griffiths.

An efficient method to overcome seed dormancy in Scotch broom ( Cytisus scoparius)

Seeds of many wild members of the Leguminosae and Solanaceae have hard seed coats which restrict water absorption by the embryo. Failure to imbibe limits O 2 to the embryo and leaching of inhibitors, therein effectively enforcing dormancy of the embryo. For applied uses, dormancy-breaking treatments are required to provide more uniform and rapid seed germination responses. Permeability may be improved by scarifying the seed coat by mechanical means (e.g. clipping, abrasion or immersion in hot water) or chemically with strong oxidative agents (e.g. sulfuric acid, sodium hypochlorite). In either case, results obtained are often less than satisfactory and commonly a fraction of the seeds are damaged, significantly reducing the overall viability of the seed lot. An improved method of propagation from seed is desirable. We report here a simple, yet effective means to overcome dormancy in Scotch broom, a plant with potential value in dune erosion control. Sequential, rapid immersion in hot water followed by liquid nitrogen dramatically improved seed imbibition and germination responses by as much as 3.5 fold. The most effective immersion times and sequence of pretreatments were identified. The strategy outlined in this paper may be widely applicable to improving seed propagation of recalcitrant species.