Settling of fixed erythrocyte suspension droplets

The fractionation of micron-size particles according to physical properties of size, density and surface characteristics by centrifugation and electrophoresis is hindered when the particles behave collectively rather than individually. The formation and sedimentation of droplets containing particles is an extreme example of collective behavior and a major problem for these separation methods when large quantities of particles need to be fractionated. In this paper, experiments that measured droplet sizes and settling rates for a variety of particles and droplets are described. Expressions are developed relating the particle concentration in a drop to measurable quantities of the fluids and particles. The number of particles in each droplet was then estimated along with the effective droplet density and certain trends are noted. Since a major application of this work is the purification of biological cells in the range of 10 microns, for which monodisperse inert particles are not available, red blood cells from different animals fixed in glutaraldehyde provided model particle groups with the necessary size range, visibility and stability for these fluid dynamical studies.     

Transport characteristics of expiratory droplets and droplet nuclei in indoor environments with different ventilation airflow patterns

Expiratory droplets and droplet nuclei can be pathogen carriers for airborne diseases. Their transport characteristics were studied in detail in two idealized floor-supply-type ventilation flow patterns: Unidirectional-upward and single-side-floor, using a multiphase numerical model. The model was validated by running interferometric Mie imaging experiments using test droplets with nonvolatile content, which formed droplet nuclei, ultimately, in a class-100 clean-room chamber. By comparing the droplet dispersion and removal characteristics with data of two other ceiling-supply ventilation systems collected from a previous work, deviations from the perfectly mixed ventilation condition were found to exist in various cases to different extent. The unidirectional-upward system was found to be more efficient in removing the smallest droplet nuclei (formed from 1.5 mum droplets) by air extraction, but it became less effective for larger droplets and droplet nuclei. Instead, the single-side-floor system was shown to be more favorable in removing these large droplets and droplet nuclei. In the single-side-floor system, the lateral overall dispersion coefficients for the small droplets and nuclei (initial size 相似文献   

Morphometric evaluation of lipid droplet associations with secretory vesicles,mitochondria and other components in the lactating cell

The size, cellular location, and identity of surface-associated components were determined for lipid droplets in lactating cells. Transmission electron-microscopic measurements were made involving 3801 droplets in approximately 211 cells from three rats and 1197 droplets in 66 cells from a mouse. For the purposes of droplet evaluation, cells were divided into seven locations ranging from basal to secreting positions. Droplets were also categorized with respect to contact with other droplets, basolateral plasma membrane, mitochondria, Golgi apparatus, secretory vesicles, and endoplasmic reticulum-cytoplasm (ERC). Data on droplet size showed that droplet growth occurs mainly in the secretory position, confirming previously published findings. Lipid droplets from mouse tissue, although somewhat smaller in size showed similar growth trends to those of the rat. Data on numbers of droplet contacts and percentages of droplet circumferences involved in associations with other cell components showed that the dominant interaction of lipid droplets was with the ERC. However, intimate association of droplets with mitochondria was noted in all cellular locations. In addition, nursed animals exhibited a greater proportion of droplet surface association with secretory vesicles and less in contact with mitochondria in comparison to those not nursed. The significance of these relationships to milk synthesis and secretion is discussed.     

Retinyl esters form lipid droplets independently of triacylglycerol and seipin

Lipid droplets store neutral lipids, primarily triacylglycerol and steryl esters. Seipin plays a role in lipid droplet biogenesis and is thought to determine the site of lipid droplet biogenesis and the size of newly formed lipid droplets. Here we show a seipin-independent pathway of lipid droplet biogenesis. In silico and in vitro experiments reveal that retinyl esters have the intrinsic propensity to sequester and nucleate in lipid bilayers. Production of retinyl esters in mammalian and yeast cells that do not normally produce retinyl esters causes the formation of lipid droplets, even in a yeast strain that produces only retinyl esters and no other neutral lipids. Seipin does not determine the size or biogenesis site of lipid droplets composed of only retinyl esters or steryl esters. These findings indicate that the role of seipin in lipid droplet biogenesis depends on the type of neutral lipid stored in forming droplets.     

Proteomic profiling of lipid droplet-associated proteins in primary adipocytes of normal and obese mouse

Lipid droplets in adipocytes serve as the principal long-term energy storage depot of animals. There is increasing recognition that lipid droplets are not merely a static neutral lipid storage site, but in fact dynamic and multi-functional organelles. Structurally, lipid droplet consists of a neutral lipid core surrounded by a phospholipid monolayer and proteins embedded in or bound to the phospholipid layer. Proteins on the surface of lipid droplets are crucial to droplet structure and dynamics. To understand the lipid droplet-associated proteome of primary adipocyte with a large central lipid droplet, lipid droplets of white adipose tissue from C57BL/6 mice were isolated. And the proteins were extracted and analyzed by liquid chromatography coupled with tandem mass spectrometry. A total of 193 proteins including 73 previously unreported proteins were identified. Furthermore, the isotope-coded affinity tags (ICAT) was used to compare the difference of lipid droplet-associated proteomes between the normal lean and the high-fat diet-induced obese C57BL/6 mice. Of 23 proteins quantified by ICAT analysis, 3 proteins were up-regulated and 4 proteins were down-regulated in the lipid droplets of adipose tissue from the obese mice. Importantly, two structural proteins of lipid droplets, perilipin A and vimentin, were greatly reduced in the lipid droplets of the adipose tissue from the obese mice, implicating reduced protein machinery for lipid droplet stability.     

Computer modelling of the meteorological and spraying parameters that influence the aerial dispersion of agrochemical sprays

 An insight into the nature of prevailing meteorological conditions and the manner in which they interact with spraying parameters is an important prerequisite in the analysis of the dynamics of agrochemical sprays. Usually, when these sprays are projected from hydraulic nozzles, their initial velocity is greater than that of the ambient wind speed. The flowfield therefore experiences changes in speed and direction which are felt upstream as well as downstream of the spray droplets. The pattern of the droplet flow, i.e. the shape of the streamlines marking typical trajectories, will be determined by a balance of viscous forces related to wind speed, inertial forces resulting from the acceleration of the airstream and pressure forces which can be viewed in terms of the drag forces exerted on the spray droplets themselves. At a certain distance in the ensuing motion, when the initial velocity of the spray droplets has decreased sufficiently for there to be no acceleration, their trajectories will be controlled entirely by the random effects of turbulence. These two transport processes in the atmosphere can be modelled mathematically using computers. This paper presents a model that considers the velocity of spray droplets to consist of a ballistic velocity component superimposed by a random-walk velocity component. The model is used to study the influence of meteorological and spraying parameters on the three-dimensional dynamics of spray droplets projected in specified directions in neutral and unstable weather conditions. The ballistic and random-walk velocity components are scaled by factors of (1–ξ) and ξ respectively, where ξ is the ratio of the sedimentation velocity and the relative velocity between the spray droplets and the surrounding airstream. This ratio increases progressively as the initial velocity of the spray droplet decreases with air resistance and attains a maximum when the sedimentation velocity has been reached. As soon as this occurs, the random-walk process predominates. The computed effects of the release height of spray droplets, atmospheric turbulence intensity, evaporation, drop size spectrum, wind velocity and wind direction on the transport process have been studied and an analysis of spray drift is provided. Received: 5 March 1997 / Accepted: 17 December 1997     

The surface of lipid droplets is a phospholipid monolayer with a unique Fatty Acid composition

We found that caveolin-2 is targeted to the surface of lipid droplets (Fujimoto, T., Kogo, H., Ishiguro, K., Tauchi, K., and Nomura, R. (2001) J. Cell Biol. 152, 1079-1085) and hypothesized that the lipid droplet surface is a kind of membrane. To elucidate the characteristics of the lipid droplet surface, we isolated lipid droplets from HepG2 cells and analyzed them by cryoelectron microscopy and by mass spectrometry. By use of cryoelectron microscopy at the stage temperature of 4.2 K, the lipid droplet surface was observed as a single line without any fixation or staining, indicating the presence of a single layer of phospholipids. This result appeared consistent with the hypothesis that the lipid droplet surface is derived from the cytoplasmic leaflet of the endoplasmic reticulum membrane and may be continuous to it. However, mass spectrometry revealed that the fatty acid composition of phosphatidylcholine and lysophosphatidylcholine in lipid droplets is different from that of the rough endoplasmic reticulum. The ample presence of free cholesterol in lipid droplets also suggests that their surface is differentiated from the bulk endoplasmic reticulum membrane. On the other hand, although caveolin-2beta and adipose differentiation-related protein, both localizing in lipid droplets, were enriched in the low density floating fraction, the fatty acid composition of the fraction was distinct from lipid droplets. Collectively, the result indicates that the lipid droplet surface is a hemi-membrane or a phospholipid monolayer containing cholesterol but is compositionally different from the endoplasmic reticulum membrane or the sphingolipid/cholesterol-rich microdomain.     

Label-free imaging of lipid-droplet intracellular motion in early Drosophila embryos using femtosecond-stimulated Raman loss microscopy

Lipid droplets are complex organelles that exhibit highly dynamic behavior in early Drosophila embryo development. Imaging lipid droplet motion provides a robust platform for the investigation of shuttling by kinesin and dynein motors, but methods for imaging are either destructive or deficient in resolution and penetration to study large populations of droplets in an individual embryo. Here we report real-time imaging and quantification of droplet motion in live embryos using a recently developed technique termed "femtosecond-stimulated Raman loss" microscopy. We captured long-duration time-lapse images of the developing embryo, tracked single droplet motion within large populations of droplets, and measured the velocity and turning frequency of each particle at different apical-to-basal depths and stages of development. To determine whether the quantities for speed and turning rate measured for individual droplets are sufficient to predict the population distributions of droplet density, we simulated droplet motion using a velocity-jump model. This model yielded droplet density distributions that agreed well with experimental observations without any model optimization or unknown parameter estimation, demonstrating the sufficiency of a velocity-jump process for droplet trafficking dynamics in blastoderm embryos.     

Deposition of lignin droplets produced during dilute acid pretreatment of maize stems retards enzymatic hydrolysis of cellulose

Electron microscopy of lignocellulosic biomass following high-temperature pretreatment revealed the presence of spherical formations on the surface of the residual biomass. The hypothesis that these droplet formations are composed of lignins and possible lignin carbohydrate complexes is being explored. Experiments were conducted to better understand the formation of these "lignin" droplets and the possible implications they might have on the enzymatic saccharification of pretreated biomass. It was demonstrated that these droplets are produced from corn stover during pretreatment under neutral and acidic pH at and above 130 degrees C, and that they can deposit back onto the surface of residual biomass. The deposition of droplets produced under certain pretreatment conditions (acidic pH; T > 150 degrees C) and captured onto pure cellulose was shown to have a negative effect (5-20%) on the enzymatic saccharification of this substrate. It was noted that droplet density (per unit area) was greater and droplet size more variable under conditions where the greatest impact on enzymatic cellulose conversion was observed. These results indicate that this phenomenon has the potential to adversely affect the efficiency of enzymatic conversion in a lignocellulosic biorefinery.     

Sterol carrier protein-2 expression modulates protein and lipid composition of lipid droplets

Despite the critical role lipid droplets play in maintaining energy reserves and lipid stores for the cell, little is known about the regulation of the lipid or protein components within the lipid droplet. Although immunofluorescence of intact cells as well as Western analysis of isolated lipid droplets revealed that sterol carrier protein-2 (SCP-2) was not associated with lipid droplets, SCP-2 expression significantly altered the structure of the lipid droplet. First, the targeting of fatty acid and cholesterol to the lipid droplets was significantly decreased. Second, the content of several proteins important for lipid droplet function was differentially increased (perilipin A), reduced severalfold (adipose differentiation-related protein (ADRP), vimentin), or almost completely eliminated (hormone-sensitive lipase and proteins >93 kDa) in the isolated lipid droplet. Third, the distribution of lipids within the lipid droplets was significantly altered. Double labeling of cells with 12-(N-methyl)-N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-octadecanoic acid (NBD-stearic acid) and antisera to ADRP showed that 70, 24, and 13% of lipid droplets contained ADRP, NBD-stearic acid, or both, respectively. SCP-2 expression decreased the level of ADRP in the lipid droplet but increased the proportion wherein ADRP and NBD-stearic acid colocalized by 3-fold. SCP-2 expression also decreased the lipid droplet fatty acid and cholesterol mass (nmol/mg protein) by 5.2- and 6.6-fold, respectively. Finally, SCP-2 expression selectively altered the pattern of esterified fatty acids in favor of polyunsaturated fatty acids within the lipid droplet. Displacement studies showed differential binding affinity of ADRP for cholesterol and fatty acids. These data suggested that SCP-2 and ADRP play a significant role in regulating fatty acid and cholesterol targeting to lipid droplets as well as in determining their lipid and protein components.     

Molecular dynamics simulation for shape change of water-in-oil droplets

We performed molecular dynamics (MD) simulations of water-in-oil droplet shape transformations induced by the addition of polymer chains. In a prior experiment, transformations of spherical droplets to rod-like, worm-like and network-like droplets were observed. In our previous study, we reproduced rod-like droplets via coarse-grained MD simulations, and the mechanism for the droplet shape change was elucidated by considering the contact area between the chains and the surfactant head groups. However, in that simulation model, we could not reproduce the worm-like and network-like droplets. In this study, we improved the simulation model. For a small number of chains, several spherical droplets were obtained. As the number of chains increased, the spherical droplets were transformed to rod-like, worm-like and network-like shapes by coalescence of the droplets. The calculated and experimental results agreed well, and we verified that the mechanism for the droplet shape transformations observed in the present simulations could be explained by the mechanism suggested in the previous study.     

Studies on Mechanisms of Splash Dispersal of Spores, using Pseudocercosporella herpotrichoides Spores

Simulated raindrops, 4 or 5 mm in diameter, fell 13 m onto target water films, with Pseudocercosporella herpotrichoides spores incorporated into either drops or targets. Resulting splash droplets were collected on fixed photographic film and numbers of droplets, spore-carrying droplets and spores determined.
The patterns of dispersal of splash droplets, spore-carrying droplets and spores with distance and droplet size were similar for 4 mm and 5 mm incident drops with spores incorporated into either targets or drops. Numbers of droplets, spore-carrying droplets and spores decreased with increasing distance from targets and none were collected at 1 m. However, more spores were dispersed by 5 mm than by 4 mm drops and with spores in targets than with spores in incident drops. Whereas most splash droplets were in the smallest size category (0–100 μm), most spore-carrying droplets were 200–400 μm and most spores were in droplets with diameter greater than 1000 μm. Regressions of square root (number of spores) on droplet diameter were significant (p < 0.001) in all cases. The slopes of regression lines were greater when spores were in targets than when they were in incident drops. Splash droplets were collected up to a height of 70 cm, with most between 15 and 20 cm. The dye experiment showed that most splash droplets contained liquid from both incident drop and target film.     

网粒体超疏水性在茶小绿叶蝉防杀虫剂渗透中的屏障效应

【目的】茶小绿叶蝉Empoasca onukii体表覆盖的网粒体具有超疏水性，杀虫剂喷雾触碰虫体后药滴动态是否受网粒体影响尚未完全清楚。本研究旨在明确网粒体在茶小绿叶蝉成虫抵御杀虫剂雾滴渗透的屏障作用。【方法】以罗丹明B(RhB)作为指示剂添加到测试的杀虫剂(联苯菊酯和茚虫威)中，利用可拍照显微镜观察记录联苯菊酯(1.25 mg/L和0.05 mg/L)和茚虫威(0.006 mg/L和0.0009 mg/L)喷雾处理后24 h，茶小绿叶蝉成虫翅面药滴滚落、蒸发、被抖动扫除等行为动态，分析翅面药滴大小与蒸发后固化形态的关系；测定网粒体移除前后药滴与翅面的接触角，统计不同疏水性翅面上的网粒体分布密度；收集并利用扫描电镜分析叶蝉体表抖落的药滴及药剂颗粒是否含有网粒体，同时观察网粒体与翅面残留溶质接触的显微形态。【结果】药滴动态观察显示，圆球状药滴在茶小绿叶蝉成虫翅面不会自行滚落，72.0%成虫静止等待翅面药滴蒸发，蒸发后形成药剂颗粒或不规则药斑与药滴大小无关，而与虫体翅面的疏水类型有关，蒸发后24 h内翅面的药剂颗粒都被叶蝉抖动扫除；在叶蝉疏水性强翅面上，药滴的静态接触角为141.63±8.06°，药滴蒸发后形成药剂颗粒，网粒体分布密度为6.1±1.2粒/μm²，而疏水性弱的翅面上药滴蒸发形成药斑，网粒体分布密度为2.2±0.9粒/μm²；SEM图片显示被茶小绿叶蝉抖落的药滴和药剂颗粒表面均带有网粒体，药斑和药剂颗粒的显微结构显示网粒体出现聚集并与残留溶质相融合。【结论】超疏水性网粒体的均匀分布决定药滴触碰茶小绿叶蝉成虫翅面后形成圆球状，网粒体的亲油性及团聚性促使药滴蒸发后形成药剂颗粒，网粒体的脱落性使药剂颗粒可被茶小绿叶蝉成虫抖动扫除。     

Hand-to-hand transmission of herpes simplex virus type 1

Droplets of tissue culture fluid containing herpes simplex virus type 1 were placed on the palm of the hand. Each 0.01 ml droplet was taken from a stock virus suspension with a titre of 10(7.5) TCID50/0.1 ml. At 0, 15, 30, 60 and 120 min a droplet was firmly touched with the middle finger of the right hand, after which, attempts were made to recover virus from the finger. At 0 min, when the virus-containing droplet was in a liquid state, there was a 100% rate of virus recovery. By 15 min the droplets had dried out, and after touching dried out droplets there was a 40% virus recovery rate, even though experimental procedures demonstrated that infectious virus was present in the dried out droplets at all test times. If the finger was moistened with tap water or saliva, there was a 100% recovery rate of virus after touching dried out droplets, as well as after touching droplets in a liquid state.     

Surface features of the lipid droplet mediate perilipin 2 localization

All eukaryotic organisms store excess lipid in intracellular lipid droplets. These dynamic structures are associated with and regulated by numerous proteins. Perilipin 2, an abundant protein on most lipid droplets, promotes neutral lipid accumulation in lipid droplets. However, the mechanism by which perilipin 2 binds to and remains anchored on the lipid droplet surface is unknown. Here we identify features of the lipid droplet surface that influence perilipin 2 localization. We show that perilipin 2 binding to the lipid droplet surface requires both hydrophobic and electrostatic interactions. Reagents that disrupt these interactions also decrease binding. Moreover, perilipin 2 binding does not depend on other lipid droplet-associated proteins but is influenced by the lipid composition of the surface. Perilipin 2 binds to synthetic vesicles composed of dioleoylphosphatidylcholine, a phospholipid with unsaturated acyl chains. Decreasing the temperature of the binding reaction, or introducing phospholipids with saturated acyl chains, decreases binding. We therefore demonstrate a role for surface lipids and acyl chain packing in perilipin 2 binding to lipid droplets. The ability of the lipid droplet phospholipid composition to impact protein binding may link changes in nutrient availability to lipid droplet homeostasis.     

Trajectory of aerosol droplets from a sprayed bacterial suspension.

Simulated droplet trajectories of a polydispersed microbial aerosol in a laminar air flow regimen were compared with observed dispersal patterns of aerosolized Bacillus subtilis subsp. niger spores in quasilaminar airflow. Simulated dispersal patterns could be explained in terms of initial droplet sizes and whether the droplets evaporated to residual aeroplanktonic size before settling to the ground. For droplets that evaporated prior to settling out, a vertical downwind size fractionation is predicted in which the microbial residue of the smallest droplets settles the least, and is found in the airstream at about sprayer height, and progressively larger droplet residues settle to progressively lower heights. Observations of spore particle size distributions downwind from a spray source support the simulation. Droplet and particle size distributions near the source had three size fractions: one containing large, presumably nonevaporated droplets of greater than or equal to 7 microns in diameter, and two smaller fractions, with diameters of 2 to 3 microns (probably the residue of droplets containing more than one spore) and 1 to 2 microns (probably the residue from single-spore droplets). As predicted by the simulation, the aerosol settled and progressed downwind, with the number of small droplets and particles increasing in proportion to the height and distance downwind.     

Trajectory of aerosol droplets from a sprayed bacterial suspension

Simulated droplet trajectories of a polydispersed microbial aerosol in a laminar air flow regimen were compared with observed dispersal patterns of aerosolized Bacillus subtilis subsp. niger spores in quasilaminar airflow. Simulated dispersal patterns could be explained in terms of initial droplet sizes and whether the droplets evaporated to residual aeroplanktonic size before settling to the ground. For droplets that evaporated prior to settling out, a vertical downwind size fractionation is predicted in which the microbial residue of the smallest droplets settles the least, and is found in the airstream at about sprayer height, and progressively larger droplet residues settle to progressively lower heights. Observations of spore particle size distributions downwind from a spray source support the simulation. Droplet and particle size distributions near the source had three size fractions: one containing large, presumably nonevaporated droplets of greater than or equal to 7 microns in diameter, and two smaller fractions, with diameters of 2 to 3 microns (probably the residue of droplets containing more than one spore) and 1 to 2 microns (probably the residue from single-spore droplets). As predicted by the simulation, the aerosol settled and progressed downwind, with the number of small droplets and particles increasing in proportion to the height and distance downwind.     

Lipid droplet de novo formation and fission are linked to the cell cycle in fission yeast

Cells sequester neutral lipids in bodies called lipid droplets. Thus, the formation and breakdown of the droplets are important for cellular metabolism; unfortunately, these processes are difficult to quantify. Here, we used time-lapse confocal microscopy to track the formation, movement and size changes of lipid droplets throughout the cell cycle in fission yeast Schizosaccharomyces pombe. In theory, the number of lipid droplets in these cells must increase for daughter cells to have the same number of droplets as the parent at a reference point in the cell cycle. We observed stable droplet formation events in G2 phase that were divided evenly between de novo formation of nascent droplets and fission of preexisting droplets. The observations that lipid droplet number is linked to the cell cycle and that droplets can form via fission were both new discoveries. Thus, we scrutinized each fission event for multiple signatures to eliminate possible artifacts from our microscopy. We augmented our time-lapse confocal microscopy with electron microscopy, which showed lipid droplet 'intermediates': droplets shaped like dumbbells that are potentially in transition states between two spherical droplets. Using these complementary microscopy techniques and also dynamic simulations, we show that lipid droplets can form by fission.     

Heat shock protein 70 is translocated to lipid droplets in rat adipocytes upon heat stimulation

In mammalian cells, lipid storage droplets contain a triacylglycerol and cholesterol ester core surrounded by a phospholipid monolayer into which a number of proteins are imbedded. These proteins are thought to be involved in modulating the formation and metabolic functions of the lipid droplet. In this study, we show that heat stress upregulates several heat shock proteins (Hsps), including Hsp27, Hsp60, Hsp70, Hsp90, and Grp78, in primary and differentiated adipocytes. Immunostaining and immunoblotting data indicate that among the Hsps examined, only Hsp70 is induced to redirect to the lipid droplet surface in heat-stressed adipocytes. The thermal induction of Hsp70 translocation to lipid droplet does not typically happen in a temperature- or time-dependent manner and occurs abruptly at 30-40 min and rapidly achieves a steady state within 60 min after 40 degrees C stress of adipocytes. Though Hsp70 is co-localized with perilipin on the lipid droplets in stressed adipocytes, immunoprecipitation experiments suggest that Hsp70 does not directly interact with perilipin. Alkaline treatments indicate that Hsp70 associates with the droplet surface through non-hydrophobic interactions. We speculate that Hsp70 might noncovalently associate with monolayer microdomains of the lipid droplet in a manner similar to its interaction with lipid bilayer moieties composed of specific fatty acids. As an acute and specific cellular response to the heat stimulation, accumulation of Hsp70 on adipocytes lipid droplets might be involved in stabilizing the droplet monolayer, transferring nascent proteins to the lipid droplets, or chaperoning denatured proteins on the droplet for subsequent refolding.     

Supercooling and nucleation of ice in single cells

An emulsion droplet formation procedure was employed to isolate yeast cells and, in separate experiments, human red blood cells, one from another in individual droplets, and to segregate extraneous materials catalyzing the formation of ice. Emulsification succeeded in isolating the cells and permitted the observation of the supercooling of droplets containing cells whereby each droplet was observed to nucleate ice at a temperature that depended only upon the components of the droplet. The droplet formation procedures were characterized. It was shown that the surface coatings and the carrier fluids used in the preparation of the emulsions did not act as ice nucleators. It was, in this manner, possible to study the nucleation of ice brought about by supercooling and homogeneous nucleation in the volume of the droplet or by the catalysis of nucleation on or in the cells contained in the droplets. It was shown that yeast cells and red blood cells could each be supercooled to about ?40 °C in short-term experiments. The results also revealed that yeast cells did not store for infinite times at temperatures above the observed upper limit of homogeneous nucleation. The yeast cells died at rates that were exponential functions of time at ?20, ?22.5, ?25, ?29 and ?33 °C. The temperature dependence of the death rate did not correspond to a process with a normal Arrhenius activation energy. The temperature dependence did, however, suggest a potentiated heterogeneous catalysis of ice resulting in the death of the yeast cells.