Thermal performance of quartz capillaries for vitrification

In this paper we report the thermal behavior of a new approach for vitrification. Thermal performance of traditional open pulled straws is compared with a new technique based on the combined use of quartz capillaries with slush nitrogen. This new method of vitrification achieved ultrafast cooling rates of 250,000 °C/min. As a result, a much lower concentration of cryoprotectant was needed to reach vitrification. In fact, a cryoprotectant solution typically used in oocyte slow freezing protocols was shown to remain transparent after cooling to liquid nitrogen temperatures indicating apparent “vitrification”. This approach offers a new and very promising technique for vitrification of cells using low levels of cryoprotectants.     

Effect of cooling rate,cryoprotectant and holding time at different transfer temperatures on the survival of cryopreserved cell suspension culture (Puccinellia distans (L.) Parl.)

Reflexed saltmarsh-grass suspension cultures produced by seed callus were frozen to the liquid nitrogen temperature. Cooling rates, cryoprotectants and holding times were taken as a function of transfer temperatures. The highest survival of cells (45%) was found at a freezing rate of 1°C min^-1, without cryoprotectant treatments. The cryoprotectants (proline, dimethyl sulphoxide, glycerol), used at different concentrations and transfer temperatures, increased the survival rate. The maximum value was 78% at 12.5% (w/v) of proline with –30°C transfer temperature. Considerable improvement of viability (from 0% to 95%) among the 12.5 and 15.0% (v/v) dimethyl sulphoxide cryopreserved cells was achieved by holding them at – 20°C for 10–30 min before plunging into the liquid nitrogen. A 20 min holding time at 15.0% (v/v) glycerol level and – 30°C transfer temperature significantly enhanced the viability of the explants from 42% to 92%. Plants were successfully regenerated from cells cryopreserved with proline (w/v) and dimethyl sulfoxide (v/v) levels of 12.5 and 15.0%, respectively.     

Comparison of the Antimicrobial Adhesion Potential of Human Body Fluid Glycoconjugates Using Fucose-Binding Lectin (PA-IIL) of Pseudomonas aeruginosa and Ulex europaeus Lectin (UEA-I)

Pseudomonas aeruginosa produces a fucose-binding lectin (PA-IIL) which strongly binds to human cells. This lectin was shown to be highly sensitive to inhibition by fucose-bearing human milk glycoproteins. Since the glycans of these glycoproteins mimic human cell receptors, they may function as decoys in blocking lectin-dependent pathogen adhesion to the host cells. Human saliva and seminal fluid also contain such compounds, and body fluids of individuals who are “secretors” express additional fucosylated (alpha 1,2) residues. The latter are selectively detected by Ulex europaeus lectin UEA-I. The aim of the present research was to compare the PA-IIL and UEA-I interactions with human salivas and seminal fluids of “secretors” and “nonsecretors” with those obtained with the respective milks. Using hemagglutination inhibition and Western blot analyses, we showed that PA-IIL interactions with the saliva and seminal fluid glycoproteins were somewhat weaker than those obtained with the milk and that “nonsecretor” body fluids were not less efficient than those of “secretors” in PA-IIL blocking. UEA-I, which interacted only with the “secretors” glycoproteins, was most sensitive to those of the seminal fluids.     

A fine-structural study of the freeze-preservation of plant tissue cultures

Summary Suspension culture cells of sycamore (Acer pseudoplatanus L.) and carrot (Daucus carota L.) were frozen to ultralow temperatures under rapid ( 100 °C s^–1) and slow, controlled (1 or 2 °C min^–1) rates, in the presence and absence of cryoprotective compounds. After storage at –196 °C, cells were recovered by thawing either slowly, in air at room temperature (ca. 20 °C min^–1) or rapidly, in a water bath at 40 °C (ca. 100 °C min^–1). The ultrastructure of the thawed cells was examined by thin-sectioning and compared with unfrozen controls and cells examined in the frozen state. Cells frozen rapidly, in the presence of cryoprotectants, or frozen slowly in their absence, suffered serious ultrastructural damage and a total loss of viability. Carrot cells frozen at a rate of 2 °C min^–1 in the presence of cryoprotectants and thawed at either rate, yielded up to 70% of viable cells. The recovered aggregates of carrot cells comprised some centrally located, seriously damaged cells and, at the periphery, groups of cells with a high electron opacity neighbouring well preserved cells, showing little ultrastructural modification compared with unfrozen controls. The highest rate of survival of sycamore cells (ca. 30%) was observed when they were frozen at a rate of 1 °C min^–1 and thawed rapidly. In all recoverd cells of sycamore some ultrastructural modifications were evident. These included: dilation of mitochondria, plastids, golgi and ER cisternae and the nuclear envelope, decrease in polysomes, increase in nuclear and cytoplasmic microfilaments and changes in nuclear and nucleolar granularity. The probable causes and timing of the ultrastructural changes and their effects on the potential for regrowth of the recovered cells are discussed.     

“Harpoon” Model for Cell–Cell Adhesion and Recognition of Target Cells by the Natural Killer Cells

The mechanism of recognition by natural killer (NK) cells is still unknown. A dynamic model is formulated describing recognition or NK-sensitive target cells (TCs) by NK cells of NK-like cells. This model does not assume the presence of the specific NK-receptor(s) on the membrane of NK cells and corresponding specific ligands on the NK-sensitive TCs. We suggest: (1) the expression of various kinds of “non-NK receptors” and corresponding ligands (counter-receptors) on the plasma membrane of the same NK cell and, possibly, of TCs (e.g. LFA-1 and ICAM-1-ICAM3, CD2 and LFA-3; receptors for TNF and corresponding ligand etc.); (2) the presence of multiple disorders in the organization of “extracellular matrix-surface membrane-submembrane cytoskeleton” assembly of the NK-sensitive TCs; (3) non-specific primary linking of NK cell with TCs, which induces a transfer of vesicles or membrane fragments from the NK surface to the target cell surface (and perhaps vice versa). These processes may also permit the transfer of many types of receptor and counter-receptor molecules from the surface of one conjugated cell to another by vesicles or membrane fragments. After transferral through the intercellular cleft, the free receptors and counter-receptors will be localized on both cell surfaces at the contact region between conjugated cells. By this model the NK cell can “harpoon” the TC and enhance the binding forces between cells up to the critical level and then switch on killing mechanisms for the TC. By means of this “harpoon” model of cell recognition, it seems possible to explain the nature of the wide polymorphism of TCs which are sensitive to the effect of NK and NK-like cells. A mathematical model of the NK cell cytotoxic reaction is described. The model describes many nonlinear peculiarities of the cytotoxic process and predicts some new phenomena. We suggest new approaches of manipulation of cell membranes which can transform NK-resistant target cells in NK sensitive cells and vice versa.     

Effects of prostaglandin E1 and isoproterenol on cyclic AMP content of human fibroblasts modified by time and cell density in subculture

In confluent cultures of “young” (< 30 generations) human fibroblasts, maximally effective concentrations of prostaglandin E₁ (5.6 μM) and isoproterenol (2 μM) increased cyclic AMP content several hundred-fold and approximately 30-fold, respectively. On the first day after initiation of cultures at either low (approx. 3 · 10⁵ cells) or high (approx. s · 10⁶ cells) cell density the magnitude of the isoproterenol effect was similar to that in confluent cultures. It increased during the next few days, reaching a maximum around day 2–3, and then declined. On any day during the period of subculture, the magnitude of the isoproterenol effect was inversely related to cell density. Alterations in response to prostaglandin E₁ as a function of time in subculture or cell density were less dramatic. The effects of prostaglandin E₁ were, however, smaller at some point during the first few days of subculture than after day 7, and when effects of prostaglandin E₁ were minimal, those of isoproterenol were maximal and approached those of prostaglandin E₁. On any day of subculture, cells in cultures of higher density tended to accumulate more cyclic AMP in response to prostaglandin E₁ than did those in low density cultures. The effects of prostaglandin E₁ and isoproterenol on cyclic AMP content were qualitatively similar in “young” and in “old” (> 60 generations in culture) human fibroblasts although the changes associated with duration of subculture and cell density tended to be less marked with “old” cells. In the “young” fibroblasts responsiveness to isoproterenol and prostaglandin E₁ appears to correlate with cell morphology and with the fractional rate of growth in subcultures. It is suggested that the capacities of the fibroblasts to respond to these two agents may be altered independently during growth of human fibroblasts.     

Isolation and Characterization of a Composite Plasmid Rms201 Mutant Temperature Sensitive for Replication

A mutant temperature-sensitive for R-plasmid replication, Rms201ts14, was isolated from composite plasmid Rms201 after mutagenesis of P1 transducing lysate with 100 mM hydroxylamine for 40 h at 37°C. When Escherichia coli ML1410(Rms201ts14)⁺ was grown at temperatures between 40 and 42°C in L broth, antibiotic-sensitive cells were segregated. When the incubation temperature of ML1410(Rms201ts14)⁺ in L-broth was shifted to 42 from 30°C, the increase in the number of antibiotic-resistant cells ceased 90 min after the temperature shift. However, the total number of cells continuously increased, and only 3% of the cells retained the plasmid at 5 h after the temperature shift to 42°C. At 30°C the amounts of covalently closed circular deoxyribonucleic acid per chromosome of Rms201ts14 and Rms201 were 3.8 and 6.3%, respectively. Incorporation of radioactive thymidine into the covalently closed circular deoxyribonucleic acid of Rms201ts14 did not take place at 42°C, whereas radioactive thymidine was incorporated into the covalently closed circular deoxyribonucleic acid of Rms201 at a rate of 4%/chromosome even at 42°C. The synthesis of plasmid covalently closed circular deoxyribonucleic acid in a cell harboring Rms201ts14 was almost completely blocked at 42°C. These results indicated that the gene(s) responsible for plasmid deoxyribonucleic acid replication was affected in the mutant Rms201ts14. Temperature-sensitive miniplasmid pMSts214, which has a molecular weight of 5.3 × 10⁶ and encodes ampicillin resistance, was isolated from Rms201ts14. Similarly, miniplasmid pMS201, which encodes single ampicillin resistance, was isolated from its parent, Rms201, and its molecular weight was 4.7 × 10⁶. These results indicate that the gene(s) causing temperature sensitivity for replication of Rms201 resides on the miniplasmid.     

Cell cycle variation associated with feeder effects in cultures of Chinese hamster fibroblasts

Sub-confluent monolayer cultures of an established line of Chinese hamster fibroblast (Don) are shown to exhibit a density-dependent stimulation of growth. Evidence is presented that both long and short range ‘feeder effects’ are involved. Using the technique of autoradiography, cell cycle parameters have been studied in sub-confluent cultures seeded at different densities to identify the source of this density-dependent variation in growth rate. The durations of S phase, G2, and mitosis are constant as indicated by “percentage labelled mitoses” curves. A simple procedure has been developed for measurement of the fraction of a cell population in the G1 state, and this fraction is shown to be inversely related to the density of the culture. It is concluded that regulation of cell growth associated with feeder effects in cultured Don cells occurs within the G1 state. The data obtained from “percentage labelled mitoses” curves are shown to be highly consistent with the predictions of the Transition Probability model for cell cycle regulation.     

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.     

The effect of macrofixation on derepressed sugar transport systems of chick embryo fibroblasts

The large molecular weight polyaldehyde (macrofixative) obtained by periodate oxidation of dextran has been shown to react with the external cell surface. Chick embryo fibroblasts (CEF), which had been treated with macrofixative (MFx), were examined for the effect on the rates of sugar transport. The low “basal” rate of sugar uptake, seen in confluent (high density) cultures of CEF was unaffected by such treatment. Low density (rapidly growing) cultures, oncogenically transformed cultures, and glucose-starved cultures have rates of sugar uptake that are significantly higher than the “basal” level. Macrofixation was found to inhibit the induction of higher rate of glucose transport under all of these conditions. The results indicate that a difference may exist between the sugar transport system in resting (confluent) cells, and that in “derepressed” cells.     

Growth control in HeLa cells by serum and anchorage

HeLa-S3 cells in suspension cultures arrest in the G1(G0) phase of the cell cycle because DNA synthesis is controlled by serum factor(s). In monolayer cultures of identical cells DNA synthesis is constitutive, i.e. independent of external signals, and cells grow without restraint. These cells reversibly display “normal” or “transformed” properties depending on the culture conditions.     

Evolution of eye development in arthropods: phylogenetic aspects

The architecture of the adult arthropod visual system for many decades has contributed important character sets that are useful for reconstructing the phylogenetic relationships within this group. In the current paper we explore whether aspects of eye development can also contribute new arguments to the discussion of arthropod phylogeny. We review the current knowledge on eye formation in Trilobita, Xiphosura, Myriapoda, Hexapoda, and Crustacea. All euarthropod taxa share the motif of a proliferation zone at the side of the developing eye field that contributes new eye elements. Two major variations of this common motif can be distinguished: 1. The “row by row type” of Trilobita, Xiphosura, and Diplopoda. In this type, the proliferation zone at the side of the eye field generates new single, large elements with a high and variable cell number, which are added to the side of the eye and extend rows of existing eye elements. Cell proliferation, differentiation and ommatidial assembly seem to be separated in time but spatially confined within the precursors of the optic units which grow continuously once they are formed (intercalary growth). 2. The “morphogenetic front type” of eye formation in Crustacea + Hexapoda (Tetraconata). In this type, there is a clear temporal and spatial separation of the formation and differentiation processes. Proliferation and the initial steps of pattern formation take place in linear and parallel mitotic and morphogenetic fronts (the mitotic waves and the morphogenetic furrow/transition zone) and numerous but small new elements with a strictly fixed set of cells are added to the eye field. In Tetraconata, once formed, the individual ommatidia do not grow any more. Scutigeromorph chilopods take an intermediate position between these two major types. We suggest that the “row by row type” as seen in Trilobita, Xiphosura and Diplopoda represents the plesiomorphic developmental mode of eye formation from the euarthropod ground pattern whereas the “morphogenetic front type” is apomorphic for the Tetraconata. Our data are discussed with regard to two competing hypotheses on arthropod phylogeny, the “Tracheata” versus “Tetraconata” concept. The modes of eye development in Myriapoda is more parsimonious to explain in the Tetraconata hypothesis so that our data raise the possibility that myriapod eyes may not be secondarily reconstructed insect eyes as the prevailing hypothesis suggests.     

Cellular “bauplans”: Evolving unicellular forms by means of Julia sets and Pickover biomorphs

The universe of cellular forms has received scarce attention by mainstream neo-Darwinian views. The possibility that a fundamental trait of biological order may consist upon, or be guided by, developmental processes not completely amenable to natural selection was more akin to previous epochs of biological thought, i.e. the “bauplan” discussion. Thirty years ago, however, Lynn and Tucker studied the biological mechanisms responsible for defining organelles position inside cells. The fact that differentiated structures performing a specific function within the eukaryotic cell (i.e. mitochondrion, vacuole, or chloroplast) were occupying specific positions in the protoplasm was the observational and experimental support of the ‘morphogenetic field’ notion at the cellular level. In the present paper we study the morphogenetic field evolution yielding from an initial population of undifferentiated cells to diversified unicellular organisms as well as specialized eukaryotic cell types. The cells are represented as Julia sets and Pickover biomorphs, simulating the effect of Darwinian natural selection with a simple genetic algorithm. The morphogenetic field “defines” the locations where cells are differentiated or sub-cellular components (or organelles) become organized. It may be realized by different possibilities, one of them by diffusing chemicals along the Turing model. We found that Pickover cells show a higher diversity of size and form than those populations evolved as Julia sets. Another novelty is the way that cellular organelles and cell nucleus fill in the cell, always in dependence on the previous cell definition as Julia set or Pickover biomorph. Our findings support the existence of specific attractors representing the functional and stable form of a differentiated cell—genuine cellular bauplans. The configuration of the morphogenetic field is “attracted” towards one or another attractor depending on the environmental influences as modeled by a particular fitness function. The model promotes the classical discussions of D’Arcy Thompson and the more recent views of Waddington, Goodwin and others that consider organisms as dynamical systems that evolve through a ‘master plan’ of transformations, amenable to natural selection. Intriguingly, the model also connects with current developments on mechanobiology, highlighting the informational–developmental role that cytoskeletons may play.     

Infective Juveniles of the Entomopathogenic Nematode,Steinernema feltiae Produce Cryoprotectants in Response to Freezing and Cold Acclimation

Steinernema feltiae is a moderately freeze-tolerant entomopathogenic nematode which survives intracellular freezing. We have detected by gas chromatography that infective juveniles of S. feltiae produce cryoprotectants in response to cold acclimation and to freezing. Since the survival of this nematode varies with temperature, we analyzed their cryoprotectant profiles under different acclimation and freezing regimes. The principal cryoprotectants detected were trehalose and glycerol with glucose being the minor component. The amount of cryoprotectants varied with the temperature and duration of exposure. Trehalose was accumulated in higher concentrations when nematodes were acclimated at 5°C for two weeks whereas glycerol level decreased from that of the non-acclimated controls. Nematodes were seeded with a small ice crystal and held at -1°C, a regime that does not produce freezing of the nematodes but their bodies lose water to the surrounding ice (cryoprotective dehydration). This increased the levels of both trehalose and glycerol, with glycerol reaching a higher concentration than trehalose. Nematodes frozen at -3°C, a regime that produces freezing of the nematodes and results in intracellular ice formation, had elevated glycerol levels while trehalose levels did not change. Steinernema feltiae thus has two strategies of cryoprotectant accumulation: one is an acclimation response to low temperature when the body fluids are in a cooled or supercooled state and the infective juveniles produce trehalose before freezing. During this process a portion of the glycerol is converted to trehalose. The second strategy is a rapid response to freezing which induces the production of glycerol but trehalose levels do not change. These low molecular weight compounds are surmised to act as cryoprotectants for this species and to play an important role in its freezing tolerance.     

New Approaches for Studying the Permeability of Fish Embryos: Toward Successful Cryopreservation

This paper describes some new approaches for understanding the permeability of teleost embryos. The dechorionated zebrafish (Brachydanio rerio) was used as a model for basic studies of water and cryoprotectant permeability. These embryos are composed of two compartments, a large yolk (surrounded by the yolk syncytial layer) and differentiating blastoderm cells. Cellular water was distributed unequally in each compartment. Measurements indicated that the total water in the embryo was 74%, while the total water in the yolk was 42%, and total water in the blastoderm was 82%. The internal isosmotic value for the zebrafish embryo is unknown. However, for one-compartment modeling studies of membrane permeability, the mean Lp (±SEM) values were 0.022 ± 0.002 to 0.049 ± 0.008 μm × min⁻¹atm⁻¹at 40 mOsm (assuming this was one possible internal isosmotic value for the entire embryo) and 0.040 ± 0.004 to 0.1 ± 0.017 μm × min⁻¹atm⁻¹at 300 mOsm (assuming this was another possible internal isosmotic value for the entire embryo). When three- and six-somite embryos were placed in 1.5 and 2.0Mcryoprotectants (dimethyl sulfoxide and propylene glycol), osmometric measurements of volume changes indicated no cryoprotectant permeation. However, similar measurements with methanol revealed a small volume decrease (ca. 8%) and recovery (ca. 5%) for six-somite embryos in a 2.0Msolution. Magnetic resonance (MR) images of the spatial distribution of three cryoprotectants (dimethyl sulfoxide, propylene glycol, and methanol) demonstrated that only methanol permeated the entire embryo within 15 min. The other cryoprotectants exhibited little or no permeation into the yolk over 2.5 h. The results from MR spectroscopy and cryoprotectant microinjections into the yolk suggested that the yolk syncytial layer plays the critical limiting role for cryoprotectant permeation throughout the embryo.     

On the concept of the effective population size

The concept of the effective population size is discussed. It is shown that the “eigenvalue” and the “inbreeding” effective population sizes are in principle different, even though they have been sometimes identified in the literature. On the other hand the “eigenvalue” and “variance” effective sizes are usually both close when the latter exists. Since, however, there are many models for which a variance effective size cannot in principle exist, it seems useful to introduce the eigenvalue effective size and to examine some of its properties.     

Turbulence, watermass stratification and harmful algal blooms: an alternative view and frontal zones as “pelagic seed banks”

Watermass stratification has been considered the essential physical condition that dinoflagellates require to bloom because of their relative inability, unlike diatoms, to tolerate the elevated shear-stress associated with water-column mixing, turbulence and high velocity, coastal currents. The swimming speeds of 71 flagellate taxa, with a focus on dinoflagellates, are compared to the turbulence fields and vertical velocities that develop during representative wind conditions, upwelling and at frontal zones. The results suggest that the classical stratification–dinoflagellate bloom paradigm needs revision. Tolerance of turbulence, growth within well-mixed watermasses and survival and dispersal while entrained within current systems are well developed capacities among dinoflagellates. Their secretion of mucous, often copious during blooms, is suggested to be an environmental engineering strategy to dampen turbulence. Biophysical tolerance of turbulence by dinoflagellates is often accompanied by high swimming speeds. Motility speeds of many species exceed in situ vertical current velocities; this also allows diel migrational patterns and other motility-based behavior to persist. Species belonging to “mixing-drift” life-form assemblages can increase their swimming speeds through chain formation, which helps to compensate for the increased turbulence and vertical water-column velocities of their habitats. The ability of dinoflagellate species to tolerate the vertical velocities of offshore, frontal zones, where abundant populations often develop, suggests that fronts may serve as “pelagic seed banks”, occurring as pelagic analogues of nearshore seed beds, from which seed stock is dispersed. The different ecologies associated with the hypothesized, “pelagic seed banks” of vegetative cells and the “seed beds” of resting stage cells deposited onto sediments are discussed. There is a contradiction in the stratification–HAB paradigm: the quiescent conditions of a stratified watermass, with its characteristic nutrient-poor conditions are expected to promote stasis of the population, rather than growth and blooms. The analyses suggest that dinoflagellate blooms do not preponderate in stratified watermasses because the bloom species are biophysically intolerant of the higher velocities and turbulence of more mixed watermasses. The watermass stratification that often accompanies flagellate blooms is probably a secondary, parallel event and less essential than some other factor(s) in triggering the observed bloom.