Why is intracellular ice lethal? A microscopical study showing evidence of programmed cell death in cryo-exposed embryonic axes of recalcitrant seeds of Acer saccharinum

Background and Aims Conservation of the genetic diversity afforded by recalcitrant seeds is achieved by cryopreservation, in which excised embryonic axes (or, where possible, embryos) are treated and stored at temperatures lower than −180 °C using liquid nitrogen. It has previously been shown that intracellular ice forms in rapidly cooled embryonic axes of Acer saccharinum (silver maple) but this is not necessarily lethal when ice crystals are small. This study seeks to understand the nature and extent of damage from intracellular ice, and the course of recovery and regrowth in surviving tissues.Methods Embryonic axes of A. saccharinum, not subjected to dehydration or cryoprotection treatments (water content was 1·9 g H₂O g⁻¹ dry mass), were cooled to liquid nitrogen temperatures using two methods: plunging into nitrogen slush to achieve a cooling rate of 97 °C s⁻¹ or programmed cooling at 3·3 °C s⁻¹. Samples were thawed rapidly (177 °C s⁻¹) and cell structure was examined microscopically immediately, and at intervals up to 72 h in vitro. Survival was assessed after 4 weeks in vitro. Axes were processed conventionally for optical microscopy and ultrastructural examination.Key Results Immediately following thaw after cryogenic exposure, cells from axes did not show signs of damage at an ultrastructural level. Signs that cells had been damaged were apparent after several hours of in vitro culture and appeared as autophagic decomposition. In surviving tissues, dead cells were sloughed off and pockets of living cells were the origin of regrowth. In roots, regrowth occurred from the ground meristem and procambium, not the distal meristem, which became lethally damaged. Regrowth of shoots occurred from isolated pockets of surviving cells of peripheral and pith meristems. The size of these pockets may determine the possibility for, the extent of and the vigour of regrowth.Conclusions Autophagic degradation and ultimately autolysis of cells following cryo-exposure and formation of small (0·2–0·4 µm) intracellular ice crystals challenges current ideas that ice causes immediate physical damage to cells. Instead, freezing stress may induce a signal for programmed cell death (PCD). Cells that form more ice crystals during cooling have faster PCD responses.     

ELECTROKINETIC PHENOMENA : XIII. A COMPARISON OF THE ISOELECTRIC POINTS OF DISSOLVED AND CRYSTALLINE AMINO ACIDS

1. Although the isoelectric points of dissolved cystine, tyrosine, and aspartic acid molecules lie at widely differing pH values, the isoelectric points of the surfaces of these substances in the crystalline state are all near pH 2.3. This was found to be true in solutions of hydrochloric acid and in acetate buffers of approximately constant ionic strength. 2. When suspended in gelatin, tyrosine and cystine crystals adsorb the protein and attain a surface identical in behavior with gelatin-coated quartz or collodion particles. 3. Aluminum ions at low concentrations reduce the electric mobilities of tyrosine crystals to zero in a manner analogous to their effect on other surfaces. 4. Alkyl benzene droplets also have their electric mobility reduced to zero at low pH values but, unlike the amino acids, a change in sign was never noticed. 5. The mobility of tyrosine crystals is independent of crystal length between 2–100µ. Below this size the mobilities are decreased. 6. These results are discussed in connection with the concept of the general definition of the isoelectric point and the behavior of certain insoluble proteins such as wool and silk fibroin.     

Detection and Isolation of Ultrasmall Microorganisms from a 120,000-Year-Old Greenland Glacier Ice Core

The abundant microbial population in a 3,043-m-deep Greenland glacier ice core was dominated by ultrasmall cells (<0.1 μm³) that may represent intrinsically small organisms or starved, minute forms of normal-sized microbes. In order to examine their diversity and obtain isolates, we enriched for ultrasmall psychrophiles by filtering melted ice through filters with different pore sizes, inoculating anaerobic low-nutrient liquid media, and performing successive rounds of filtrations and recultivations at 5°C. Melted ice filtrates, cultures, and isolates were analyzed by scanning electron microscopy, flow cytometry, cultivation, and molecular methods. The results confirmed that numerous cells passed through 0.4-μm, 0.2-μm, and even 0.1-μm filters. Interestingly, filtration increased cell culturability from the melted ice, yielding many isolates related to high-G+C gram-positive bacteria. Comparisons between parallel filtered and nonfiltered cultures showed that (i) the proportion of 0.2-μm-filterable cells was higher in the filtered cultures after short incubations but this difference diminished after several months, (ii) more isolates were obtained from filtered (1,290 isolates) than from nonfiltered (447 isolates) cultures, and (iii) the filtration and liquid medium cultivation increased isolate diversity (Proteobacteria; Cytophaga-Flavobacteria-Bacteroides; high-G+C gram-positive; and spore-forming, low-G+C gram-positive bacteria). Many isolates maintained their small cell sizes after recultivation and were phylogenetically novel or related to other ultramicrobacteria. Our filtration-cultivation procedure, combined with long incubations, enriched for novel ultrasmall-cell isolates, which is useful for studies of their metabolic properties and mechanisms for long-term survival under extreme conditions.     

Intracellular Freezing in the Infective Juveniles of Steinernema feltiae: An Entomopathogenic Nematode

Taking advantage of their optical transparency, we clearly observed the third stage infective juveniles (IJs) of Steinernema feltiae freezing under a cryo-stage microscope. The IJs froze when the water surrounding them froze at −2°C and below. However, they avoid inoculative freezing at −1°C, suggesting cryoprotective dehydration. Freezing was evident as a sudden darkening and cessation of IJs'' movement. Freeze substitution and transmission electron microscopy confirmed that the IJs of S. feltiae freeze intracellularly. Ice crystals were found in every compartment of the body. IJs frozen at high sub-zero temperatures (−1 and −3°C) survived and had small ice crystals. Those frozen at −10°C had large ice crystals and did not survive. However, the pattern of ice formation was not well-controlled and individual nematodes frozen at −3°C had both small and large ice crystals. IJs frozen by plunging directly into liquid nitrogen had small ice crystals, but did not survive. This study thus presents the evidence that S. feltiae is only the second freeze tolerant animal, after the Antarctic nematode Panagrolaimus davidi, shown to withstand extensive intracellular freezing.     

FURTHER ELECTRON MICROSCOPE STUDIES ON FIBRILLAR ORGANIZATION OF THE GROUND CYTOPLASM OF CHAOS CHAOS

Further evidence for fibrillar organization of the ground cytoplasm of Chaos chaos is presented. Fixations with osmium tetroxide at pH 6 or 8 and with glutaraldehyde at pH 6 or 7 were used on two preparations: (a) single actively streaming cells; (b) prechilled cells treated with 0.05% Alcian blue in the cold and returned to room temperature for 5–10 min. In addition, a 50,000 g pellet of homogenized cells was examined after fixation with glutaraldehyde-formaldehyde alone. In sections from actively streaming cells considerable numbers of filaments were observed in the uroid regions after glutaraldehyde fixation, whereas only traces of filaments were seen after osmium tetroxide fixation at either pH 6 or 8. Microtubules were not seen. In sections from dye-treated cells, filaments (4–6 mµ) and fibrils (12–15 mµ) were found with all three fixatives. The 50,000 g pellet was heterogeneous but contained both clumps of fibrils and single thick fibrils like those seen in the cytoplasm of dye-treated cells. Many fibrils of the same dimensions (12–15 mµ wide, 0.5 µ long) were also seen in the supernatant above the pellet. Negative staining showed that some fibrils separated into at least three strands of 4–6 mµ filaments.     

Feasibility of the Assessment of Cholesterol Crystals in Human Macrophages Using Micro Optical Coherence Tomography

The presence of cholesterol crystals is a hallmark of atherosclerosis, but until recently, such crystals have been considered to be passive components of necrotic plaque cores. Recent studies have demonstrated that phagocytosis of cholesterol crystals by macrophages may actively precipitate plaque progression via an inflammatory pathway, emphasizing the need for methods to study the interaction between macrophages and crystalline cholesterol. In this study, we demonstrate the feasibility of detecting cholesterol in macrophages in situ using Micro-Optical Coherence Tomography (µOCT), an imaging modality we have recently developed with 1-µm resolution. Macrophages containing cholesterol crystals frequently demonstrated highly scattering constituents in their cytoplasm on µOCT imaging, and µOCT was able to evaluate cholesterol crystals in cultured macrophage cells. Our results suggest that µOCT may be useful for the detection and characterization of inflammatory activity associated with cholesterol crystals in the coronary artery.     

Phylogenetic Analysis of Anaerobic Psychrophilic Enrichment Cultures Obtained from a Greenland Glacier Ice Core

The examination of microorganisms in glacial ice cores allows the phylogenetic relationships of organisms frozen for thousands of years to be compared with those of current isolates. We developed a method for aseptically sampling a sediment-containing portion of a Greenland ice core that had remained at −9°C for over 100,000 years. Epifluorescence microscopy and flow cytometry results showed that the ice sample contained over 6 × 10⁷ cells/ml. Anaerobic enrichment cultures inoculated with melted ice were grown and maintained at −2°C. Genomic DNA extracted from these enrichments was used for the PCR amplification of 16S rRNA genes with bacterial and archaeal primers and the preparation of clone libraries. Approximately 60 bacterial inserts were screened by restriction endonuclease analysis and grouped into 27 unique restriction fragment length polymorphism types, and 24 representative sequences were compared phylogenetically. Diverse sequences representing major phylogenetic groups including alpha, beta, and gamma Proteobacteria as well as relatives of the Thermus, Bacteroides, Eubacterium, and Clostridium groups were found. Sixteen clone sequences were closely related to those from known organisms, with four possibly representing new species. Seven sequences may reflect new genera and were most closely related to sequences obtained only by PCR amplification. One sequence was over 12% distant from its closest relative and may represent a novel order or family. These results show that phylogenetically diverse microorganisms have remained viable within the Greenland ice core for at least 100,000 years.     

VISUALIZATION OF FREEZING DAMAGE

Freeze-cleaving can be used as a direct probe to examine the ultrastructural alterations of biological material due to freezing. We examined the thesis that at least two factors, which are oppositely dependent upon cooling velocity, determine the survival of cells subjected to freezing. According to this thesis, when cells are cooled at rates exceeding a critical velocity, a decrease in viability is caused by the presence of intracellular ice; but cells cooled at rates less than this critical velocity do not contain appreciable amounts of intracellular ice and are killed by prolonged exposure to a solution that is altered by the presence of ice. As a test of this hypothesis, we examined freeze-fractured replicas of the yeast Saccharomyces cerevisiae after suspensions had been cooled at rates ranging from 1.8 to 75,000°C/min. Some of the frozen samples were cleaved and replicated immediately in order to minimize artifacts due to sample handling. Other samples were deeply etched or were rewarmed to -20°C and recooled before replication. Yeast cells cooled at or above the rate necessary to preserve maximal viability (~7°C/min) contained intracellular ice, whereas cells cooled below this rate showed no evidence of intracellular ice.     

Expression of a bacterial ice nucleation gene in plants

We have introduced an ice nucleation gene (inaZ) from Pseudomonas syringae pv. syringae into Nicotiana tabacum, a freezing-sensitive species, and Solanum commersonii, a freezing-tolerant species. Transformants of both species showed increased ice nucleation activity over untransformed controls. The concentration of ice nuclei detected at −10.5°C in 15 different primary transformants of S. commersonii varied by over 1000-fold, and the most active transformant contained over 100 ice nuclei/mg of tissue. The temperature of the warmest freezing event in plant samples of small mass was increased from approximately −12°C in the untransformed controls to −4°C in inaZ-expressing transformants. The threshold nucleation temperature of samples from transformed plants did not increase appreciably with the mass of the sample. The most abundant protein detected in transgenic plants using immunological probes specific to the inaZ protein exhibited a higher mobility on sodium dodecyl sulfate polyacrylamide gels than the inaZ protein from bacterial sources. However, some protein with a similar mobility to the inaZ protein could be detected. Although the warmest ice nucleation temperature detected in transgenic plants is lower than that conferred by this gene in P. syringae (−2°C), our results demonstrate that the ice nucleation gene of P. syringae can be expressed in plant cells to produce functional ice nuclei.     

Bacterial Activity at −2 to −20°C in Arctic Wintertime Sea Ice

Arctic wintertime sea-ice cores, characterized by a temperature gradient of −2 to −20°C, were investigated to better understand constraints on bacterial abundance, activity, and diversity at subzero temperatures. With the fluorescent stains 4′,6′-diamidino-2-phenylindole 2HCl (DAPI) (for DNA) and 5-cyano-2,3-ditoyl tetrazolium chloride (CTC) (for O₂-based respiration), the abundances of total, particle-associated (>3-μm), free-living, and actively respiring bacteria were determined for ice-core samples melted at their in situ temperatures (−2 to −20°C) and at the corresponding salinities of their brine inclusions (38 to 209 ppt). Fluorescence in situ hybridization was applied to determine the proportions of Bacteria, Cytophaga-Flavobacteria-Bacteroides (CFB), and Archaea. Microtome-prepared ice sections also were examined microscopically under in situ conditions to evaluate bacterial abundance (by DAPI staining) and particle associations within the brine-inclusion network of the ice. For both melted and intact ice sections, more than 50% of cells were found to be associated with particles or surfaces (sediment grains, detritus, and ice-crystal boundaries). CTC-active bacteria (0.5 to 4% of the total) and cells detectable by rRNA probes (18 to 86% of the total) were found in all ice samples, including the coldest (−20°C), where virtually all active cells were particle associated. The percentage of active bacteria associated with particles increased with decreasing temperature, as did the percentages of CFB (16 to 82% of Bacteria) and Archaea (0.0 to 3.4% of total cells). These results, combined with correlation analyses between bacterial variables and measures of particulate matter in the ice as well as the increase in CFB at lower temperatures, confirm the importance of particle or surface association to bacterial activity at subzero temperatures. Measuring activity down to −20°C adds to the concept that liquid inclusions in frozen environments provide an adequate habitat for active microbial populations on Earth and possibly elsewhere.     

Effect of alkyl alcohols on partially unfolded state of Proteinase K: Differential stability of α-helix and β-sheet rich regions of the enzyme

Proteinase K (E.C. 3.4.21.64), a serine proteinase from fungus Tritirachium album, has been used as a model system to investigate the conformational changes induced by monohydric alcohols at low pH. Proteinase K belongs to α/β class of proteins and maintains structural integrity in the range of pH 7.0–3.0. Enzyme acquires partially unfolded conformation (U_P) at pH 2.5 with lower activity, partial loss of tertiary structure and exposure of some hydrophobic patches. Proteinase K in stressed state at pH 2.5 is chosen and the conformational changes induced by alkyl alcohols (methanol/ethanol/isopropanol) are studied. At critical concentration of alcohol, conformational switch occurs in the protein structure from α/β to β-sheet driving the protein into O-state. Complete loss of tertiary contacts and proteolytic activity in O-sate emphasize the involvement of alpha regions in maintaining the active site of the enzyme. Moreover, isopropanol induced unfolding of proteinase K in U_P state occurred in two steps with the formation of β state at low alcohol concentration followed by stabilization of β state at high alcohol concentration. GuHCl and temperature induced unfolding of proteinase K in O-state (in 50% isopropanol) is non-cooperative as the transition curves are biphasic. This suggests that the structure of proteinase K in O-state has melted alpha regions and stabilized beta regions and that these differentially stabilized regions unfold sequentially. Further, the O-state of proteinase K can be attained from complete unfolded protein by the addition of 50% isopropanol. Hence the alcohol-induced O-state is different from native state or completely unfolded state and shows characteristics of the molten globule-like state. Thus, this state may be functioning as an intermediary in the folding pathway of proteinase K.     

Comparison of Disulfide Contents and Solubility at Alkaline pH of Insecticidal and Noninsecticidal Bacillus thuringiensis Protein Crystals

We compared two insecticidal and eight noninsecticidal soil isolates of Bacillus thuringiensis with regard to the solubility of their proteinaceous crystals at alkaline pH values. The protein disulfide contents of the insecticidal and noninsecticidal crystals were equivalent. However, six of the noninsecticidal crystals were soluble only at pH values of ≥12. This lack of solubility contributed to their lack of toxicity. One crystal type which was soluble only at pH ≥12 (strain SHP 1-12) did exhibit significant toxicity to tobacco hornworm larvae when the crystals were presolubilized. In contrast, freshly prepared crystals from the highly insecticidal strain HD-1 were solubilized at pH 9.5 to 10.5, but when these crystals were denatured, by either 8 M urea or autoclave temperatures, they became nontoxic and were soluble only at pH values of ≥12. These changes in toxicity and solubility occurred even though the denatured HD-1 crystals were morphologically indistinguishable from native crystals. Our data are consistent with the view that insecticidal crystals contain distorted, destabilized disulfide bonds which allow them to be solubilized at pH values (9.5 to 10.5) characteristic of lepidopteran and dipteran larval midguts.     

Survival of plant tissue at super-low temperatures v. An electron microscope study of ice in cortical cells cooled rapidly

Experiments were carried out with cortical cells in twig bark of mulberry trees in winter in order to clarify the mechanism of survival at super-low temperatures with rapid cooling and rewarming. Attention was given to the relation between the existence of intracellular ice crystals and survival.

Cortical cells were cooled rapidly by direct immersion into liquid nitrogen or isopentane cooled at various temperatures. After immersion, they were freeze-substituted with absolute ethanol at −78°. They were then embedded, sectioned and examined under the electron microscope for the presence and distribution of cavities left after ice removal.

Cells were found to remain alive and contain no ice cavities when immersed rapidly into isopentane baths kept below −60°. Those cells at intermediate temperatures from −20° to −45°, were almost all destroyed. It was also observed that many ice cavities were contained in the cells immersed rapidly into isopentane baths at −30°. The data seem to indicate that no ice crystals were formed when cooled rapidly by direct immersion into isopentane baths below −60° or into liquid nitrogen.

The tissue sections immersed in liquid nitrogen were rapidly transferred to isopentane baths at temperatures ranging from −70° to −10° before rapid rewarming. There was little damage when samples were held at temperatures below −50° for 10 minutes or below −60° for 16 hours. No cavities were found in these cells. Above −45°, and especially at −30°, however, all cells were completely destroyed even when exposed only for 1 minute. Many ice cavities were observed throughout these cells. The results obtained may be explained in terms of the growth rate of intracellular ice crystals.

     

CRYSTALLIZATION OF PEPSIN FROM ALCOHOL

1. Pepsin is soluble in 65 per cent alcohol and may be readily crystallized from 20 per cent alcohol. The crystals appear as needles or plates which may be transformed into the usual hexagonal bipyramids by recrystallization from water. The different crystals are probably two crystalline forms of the same chemical substance. 2. The enzyme is quite stable in 20 per cent alcohol at pH 2.0 but is inactivated by high concentrations of alcohol. 3. The enzyme is stable for several hours in 65 per cent alcohol at pH 4.0 to 5.0 but is rapidly inactivated in more acid solution. 4. No increase in activity could be noted after treatment with hydrogen peroxide. 5. No proteolytic activity either before or after treatment with hydrogen peroxide could be found in trichloracetic acid filtrates, butyl alcohol extracts of pepsin preparations, or oxidized phenylhydrazine solutions.     

Ice Nucleation Activity in Fusarium acuminatum and Fusarium avenaceum

Twenty fungal genera, including 14 Fusarium species, were examined for ice nucleation activity at −5.0°C, and this activity was found only in Fusarium acuminatum and Fusarium avenaceum. This characteristic is unique to these two species. Ice nucleation activity of F. avenaceum was compared with ice nucleation activity of a Pseudomonas sp. strain. Cumulative nucleus spectra are similar for both microorganisms, while the maximum temperatures of ice nucleation were −2.5°C for F. avenaceum and −1.0°C for the bacteria. Ice nucleation activity of F. avenaceum was stable at pH levels from 1 to 13 and tolerated temperature treatments up to 60°C, suggesting that these ice nuclei are more similar to lichen ice nuclei than to bacterial ones. Ice nuclei of F. avenaceum, unlike bacterial ice nuclei, pass through a 0.22-μm-pore-size filter. Fusarial nuclei share some characteristics with the so-called leaf-derived nuclei with which they might be identified: they are cell free and stable up to 60°C, and they are found in the same kinds of environment. Highly stable ice nuclei produced by fast-growing microorganisms have potential applications in biotechnology. This is the first report of ice nucleation activity in free-living fungi.     

Histochemical detection of GM1 ganglioside using cholera toxin-B subunit. Evaluation of critical factors optimal for in situ detection with special emphasis to acetone pre-extraction

A comparison of histochemical detection of GM1 ganglioside in cryostat sections using cholera toxin B-subunit after fixation with 4% formaldehyde and dry acetone gave tissue-dependent results. In the liver no pre-treatment showed detectable differences related to GM1 reaction products, while studies in the brain showed the superiority of acetone pre-extraction (followed by formaldehyde), which yielded sharper images compared with the diffuse, blurred staining pattern associated with formaldehyde. Therefore, the aim of our study was to define the optimal conditions for the GM1 detection using cholera toxin B-subunit.Ganglioside extractability with acetone, the ever neglected topic, was tested comparing anhydrous acetone with acetone containing admixture of water. TLC analysis of acetone extractable GM1 ganglioside from liver sections did not exceed 2% of the total GM1 ganglioside content using anhydrous acetone at −20°C, and 4% at room temperature. The loss increased to 30.5% using 9:1 acetone/water. Similarly, photometric analysis of lipid sialic acid, extracted from dried liver homogenates with anhydrous acetone, showed the loss of gangliosides into acetone 3.0±0.3% only. The loss from dried brain homogenate was 9.5±1.1%.Thus, anhydrous conditions (dry tissue samples and anhydrous acetone) are crucial factors for optimal in situ ganglioside detection using acetone pre-treatment. This ensures effective physical fixation, especially in tissues rich in polar lipids (precipitation, prevention of in situ diffusion), and removal of cholesterol, which can act as a hydrophobic blocking barrier.Key words: fixation, GM1 ganglioside, cholera toxin, anhydrous acetone, 4% formaldehyde.     

FORMATION OF TRYPSIN FROM TRYPSINOGEN BY AN ENZYME PRODUCED BY A MOLD OF THE GENUS PENICILLIUM

1. A powerful kinase which changes trypsinogen to trypsin was found to be present in the synthetic liquid culture medium of a mold of the genus Penicillium. 2. The concentration of kinase in the medium is increased gradually during the growth of the mold organism and continues to increase for some time even after the mold has ceased growing. 3. Mold kinase transforms trypsinogen to trypsin only in an acid medium. It differs thus from enterokinase and trypsin which activate trypsinogen best in a slightly alkaline medium. 4. The action of the mold kinase in the process of transformation of trypsinogen is that of a typical enzyme. The process follows the course of a catalytic unimolecular reaction, the rate of formation of a definite amount of trypsin being proportional to the concentration of kinase added. The ultimate amount of trypsin formed, however, is independent of the concentration of kinase used. 5. The formation of trypsin from trypsinogen by mold kinase is not accompanied by any measurable loss of protein. 6. The temperature coefficient of formation of trypsin from trypsinogen by mold kinase varies from Q _5–15 = 1.70 to Q _25–30 = 1.25 with a corresponding variation in the value of µ from 8100 to 4250. 7. Trypsin formed from trypsinogen by means of mold kinase is identical in crystalline form with the crystalline trypsin obtained by spontaneous autocatalytic activation of trypsinogen at pH 8.0. The two products have within the experimental error the same solubility and specific activity. A solution saturated with the crystals of either one of the trypsin preparations does not show any increase in protein concentration or activity when crystals of the other trypsin preparation are added. 8. The Penicillium mold kinase has a slight activating effect on chymo-trypsinogen the rate being only 1–2 per cent of that of trypsinogen. The activation, as in the case of trypsinogen, takes place only in an acid medium. 9. Mold kinase is rapidly destroyed when brought to pH 6.5 or higher, and also when heated to 70°C. In the temperature range of 50–60°C. the inactivation of kinase follows a unimolecular course with a temperature coefficient of Q ₁₀ = 12.1 and µ = 53,500. The molecular weight of mold kinase, as determined by diffusion, is 40,000.     

The validity of the cholesterol nucleation assay

The validity of the cholesterol nucleation assay rests on the assumption that all cholesterol crystals are removed at the start of the assay so that de novo formation of crystals can be studied. In this paper we have tested the validity of this assumption. Cholesterol crystals were added to supersaturated model bile. Subsequently the mixtures were either filtered over a 0.22 μm filter or centrifuged at 37°C for 2 h at 100 000 × g. After ultracentrifugation the isotropic interphase was collected. Using polarized light microscopy no crystals could be visualized in this fraction. However, the nucleation time of the isotropic interphase decreased from 6.8 ± 1.1 days to 1.8 ± 0.2 days (mean ± S.E., P < 0.01, n = 5) when 10–100 μg/ml crystals were added prior to centrifugation. Similar results were observed when instead of centrifugation the mixtures containing crystals were filtered. After filtration over a 0.22 μm filter no crystals could be detected in the filtrate. Yet the nucleation time of the filtrate decreased from 6.4 ± 0.7 days to 3.1 ± 0.5 days (mean ± S.E.) when 10 μg/ml cholesterol crystals were added before filtration (n = 10, P < 0.01). Since no cholesterol crystals could be detected at the start of the assay the reduction in nucleation time must have been brought about by cholesterol microcrystals that passed through the filter. Supplementation of cholesterol crystals to model bile did not accelerate the nucleation time when the samples were passed over a 0.02 μm filter, indicating that the size of the microcrystals was larger than 20 nm. The effect of addition of cholesterol crystals prior to filtration over a 0.22 μm filter was also tested in the crystal growth assay recently developed by Busch et al. ((1990) J. Lipid Res. 31, 1903–1909). Addition of crystals had only a minor effect on the assay. In conclusion, the reduced nucleation time of biles from gallstone patients is probably not only due to the presence of promoting or the absence of inhibiting proteins, but can be caused by the presence of small cholesterol crystals in these biles.     

Changes in Osmotic Pressure and Mucilage during Low-Temperature Acclimation of Opuntia ficus-indica

Opuntia ficus-indica, a Crassulacean acid metabolism plant cultivated for its fruits and cladodes, was used to examine chemical and physiological events accompanying low-temperature acclimation. Changes in osmotic pressure, water content, low molecular weight solutes, and extracellular mucilage were monitored in the photosynthetic chlorenchyma and the water-storage parenchyma when plants maintained at day/night air temperatures of 30/20°C were shifted to 10/0°C. An increase in osmotic pressure of 0.13 megapascal occurred after 13 days at 10/0°C. Synthesis of glucose, fructose, and glycerol accounted for most of the observed increase in osmotic pressure during the low-temperature acclimation. Extracellular mucilage and the relative apoplastic water content increased by 24 and 10%, respectively, during exposure to low temperatures. These increases apparently favor the extracellular nucleation of ice closer to the equilibrium freezing temperature for plants at 10/0°C, which could make the cellular dehydration more gradual and less damaging. Nuclear magnetic resonance studies helped elucidate the cellular processes during ice formation, such as those revealed by changes in the relaxation times of two water fractions in the chlorenchyma. The latter results suggested a restricted mobility of intracellular water and an increased mobility of extracellular water for plants at 10/0°C compared with those at 30/20°C. Increased mobility of extracellular water could facilitate extracellular ice growth and thus delay the potentially lethal intracellular freezing during low-temperature acclimation.     

Influence of Pretreatment and Experimental Conditions on Electrophoretic Mobility and Hydrophobicity of Cryptosporidium parvum Oocysts

Surface properties of Cryptosporidium parvum oocysts were investigated by using electrophoretic mobility and hydrophobicity measurements. Oocysts purified from calf feces by several sucrose flotation steps and deionized water (DI) washes (DIS method) had an electrophoretic mobility (neutral surface charge) near 0.0 m² V⁻¹ s⁻¹ over a pH range of 2 to 10. The mean electrophoretic mobility of oocysts stored in DI containing a mixture of antibiotics had a lower standard deviation (ς = 0.36) than that of oocysts stored in DI without antibiotics (ς = 0.53); their electrophoretic mobility remained unchanged up to 121 days after collection. The electrophoretic mobility of oocysts purified on a cold Percoll-sucrose gradient after the feces was defatted with ethyl acetate (EAPS method) varied linearly with pH from 0.0 m² V⁻¹ s⁻¹ at pH 2.4 to −3.2 × 10⁻⁸ m² V⁻¹ s⁻¹ at pH 10 (ς = 0.52), thus displaying the negative surface charge at neutral pH observed by other researchers. The hydrophobicity of oocysts and two types of polystyrene beads was measured as a function of ionic strength by adhesion to polystyrene. Oocysts were purified by the DIS method. The ionic strength of the suspending solution was varied from 0 to 95 mmol liter⁻¹. Two-week-old oocysts exhibited strong adhesion (~85%) at ionic strengths of 0 to 10 mmol liter⁻¹ and moderate adhesion (~20%) at ionic strengths of 20 to 95 mmol liter⁻¹. Two-month-old oocysts exhibited high adhesion (~60 to 80%) at all ionic strengths. These results show that adhesion properties governed by the electrophoretic mobility of purified C. parvum oocysts can be altered by the method of purification and that hydrophobicity can change as oocysts age.