Structural and dielectric properties of synthetic glycolipids in mixtures with water

Three synthetically produced glycolipids, N-(β-D-glucopyranosyl)-N-octadecyl-stearoylamide (OSGA), N-(β-D-glucopyranosyl-N-octadecyl-oleoylamide (OOGA), N-(β-D-galactopyranosyl)-N-octadecyl-lauroylamide (OLGA) have been studied in different mixtures with water by x-ray diffraction and dielectric measurements with microwaves at 9.4 GHz. The measurements were performed in the temperature range -50-70°C. X-Ray diffraction revealed a direct L_β' → H transition at 20°C, 60°C, and 45°C depending on the glycolipid species but nearly not on the water content. The hexagonal phases are saturated at a water content of ≈20 wt%. The lamellar phase absorbs even less water (< 10 wt%). The dielectric data show that in the H phase the binding of water is stronger than in the L_β' phase. In the temperature range below 0°C, OSGA and OOGA show a “subzero transition” due to the freeze-out of water in a separate ice phase. This transition can be seen in an abrupt decrease of the dielectric function because the dielectric response of ice is much smaller at microwave frequencies. OLGA does not show the subzero transition but an additional transition, hexagonal → distorted hexagonal at 60°C.     

BOUND WATER IN MUSCLE

1. The amount of free unfrozen water, i.e. water acting as normal solvent, in frog''s muscle at temperatures below the initial freezing-point has been calculated from the vapour pressure isotherm of the muscle. 2. Significant amounts of free water are present at –20°C. The total amount of unfrozen water at –20°C. cannot, therefore, be taken as a measure of the bound water in muscle. 3. The calculated values of free water, when compared with experimentally determined values of total unfrozen water, indicate that the amount of bound water in muscle at various temperatures is small. 4. A temperature considerably below –20°C., roughly between –40° and –60°C., is required to freeze completely the free water in muscle.     

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.     

Effect of cold acclimation on intracellular ice formation in isolated protoplasts

When cooled at rapid rates to temperatures between −10 and −30°C, the incidence of intracellular ice formation was less in protoplasts enzymically isolated from cold acclimated leaves of rye (Secale cereale L. cv Puma) than that observed in protoplasts isolated from nonacclimated leaves. The extent of supercooling of the intracellular solution at any given temperature increased in both nonacclimated and acclimated protoplasts as the rate of cooling increased. There was no unique relationship between the extent of supercooling and the incidence of intracellular ice formation in either nonacclimated or acclimated protoplasts. In both nonacclimated and acclimated protoplasts, the extent of intracellular supercooling was similar under conditions that resulted in the greatest difference in the incidence of intracellular ice formation—cooling to −15 or −20°C at rates of 10 or 16°C/minute. Further, the hydraulic conductivity determined during freeze-induced dehydration at −5°C was similar for both nonacclimated and acclimated protoplasts. A major distinction between nonacclimated and acclimated protoplasts was the temperature at which nucleation occurred. In nonacclimated protoplasts, nucleation occurred over a relatively narrow temperature range with a median nucleation temperature of −15°C, whereas in acclimated protoplasts, nucleation occurred over a broader temperature range with a median nucleation temperature of −42°C. We conclude that the decreased incidence of intracellular ice formation in acclimated protoplasts is attributable to an increase in the stability of the plasma membrane which precludes nucleation of the supercooled intracellular solution and is not attributable to an increase in hydraulic conductivity of the plasma membrane which purportedly precludes supercooling of the intracellular solution.     

Measurement of Proteolysis in Natural Waters

Microbiological proteolysis in Lake Champlain water was measured in situ and in vitro by the spectrophotometric measurement of the rate of release of soluble color from an insoluble azure dye derivative of hide powder. Water samples sterilized by microfiltration were never proteolytic. In situ proteolysis was found to be very dependent upon water temperature (1 to 23°C). No measurable activity was observed below 4°C. The in vitro proteolysis rate at 20°C was found to be 2.3 times the rate at 15°C and 6 times the rate at 10°C. Water taken from beneath the ice-covered lake throughout the winter and tested in the laboratory at 20°C was found to show an increasing proteolytic potential during the winter months. The highest activity was obtained as the ice broke up in early spring. Microbiological proteolysis in water from Burlington Harbor was often four times that found in center lake water. In most experiments proteolysis was inhibited completely by 2 μg of Cu²⁺ and inhibited 67% by 0.75 μg/ml. Proteolysis was markedly stimulated by 20 to 40 μg of Casitone or Casamino Acids per ml. The predominant bacteria growing in the proteolysis flasks were species of Pseudomonas and Flavobacterium. Pure cultures of Pseudomonas required traces of Casitone, Casamino Acids, or yeast extract for proteolysis of hide powder azure, whereas those of Flavobacterium did not. The requirement could not be met by a mixture of 21 amino acids and eight vitamins.     

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.     

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.     

Development, distribution, and characteristics of intrinsic, nonbacterial ice nuclei in prunus wood

Ice nuclei active at approximately −2°C and intrinsic to woody tissues of Prunus spp. were shown to have properties distinct from bacterial ice nuclei. Soaking 5-centimeter peach stem sections in water for 4 hours lowered the mean ice nucleation temperature to below −4°C, nearly 2°C lower than stems inoculated with ice nucleation-active Pseudomonas syringae strain B301D. Ice nucleation activity in peach was fully restored by air-drying woody stem sections for a few hours. The ice nuclei in woody tissue were inactivated between 40 and 50°C, but unaffected by treatment with bacterial ice nucleation inhibitors (i.e. NaOCl, tartaric acid, Triton XQS-20), sulfhydryl reagents (i.e. p-hydroxymercuribenzoate and iodine) and Pronase. Ice nuclei could not be dislodged from stems by sonication and were shown to be equally distributed in peach bud and internodal stem tissue on a per unit mass basis; outer and inner stem tissues were also indistinguishable in ice nucleation activity. Development of ice nuclei in immature peach and sweet cherry stems did not occur until midsummer and their formation was essentially complete by late August. Once formed the ice nuclei intrinsic to woody stems were stable and unaffected by seasonal changes in growth. The apparent physiological function of the ice nuclei is discussed in relation to supercooling and mechanisms of cold hardiness in Prunus spp.     

Intracellular ice and cell survival in cryo-exposed embryonic axes of recalcitrant seeds of Acer saccharinum: an ultrastructural study of factors affecting cell and ice structures

Background and Aims

Cryopreservation is the only long-term conservation strategy available for germplasm of recalcitrant-seeded species. Efforts to cryopreserve this form of germplasm are hampered by potentially lethal intracellular freezing events; thus, it is important to understand the relationships among cryo-exposure techniques, water content, structure and survival.

Methods

Undried embryonic axes of Acer saccharinum and those rapidly dried to two different water contents were cooled at three rates and re-warmed at two rates. Ultrastructural observations were carried out on radicle and shoot tips prepared by freeze-fracture and freeze-substitution to assess immediate (i.e. pre-thaw) responses to cooling treatments. Survival of axes was assessed in vitro.

Key Results

Intracellular ice formation was not necessarily lethal. Embryo cells survived when crystal diameter was between 0·2 and 0·4 µm and fewer than 20 crystals were distributed per μm² in the cytoplasm. Ice was not uniformly distributed within the cells. In fully hydrated axes cooled at an intermediate rate, the interiors of many organelles were apparently ice-free; this may have prevented the disruption of vital intracellular machinery. Intracytoplasmic ice formation did not apparently impact the integrity of the plasmalemma. The maximum number of ice crystals was far greater in shoot apices, which were more sensitive than radicles to cryo-exposure.

Conclusions

The findings challenge the accepted paradigm that intracellular ice formation is always lethal, as the results show that cells can survive intracellular ice if crystals are small and localized in the cytoplasm. Further understanding of the interactions among water content, cooling rate, cell structure and ice structure is required to optimize cryopreservation treatments without undue reliance on empirical approaches.     

Frost injury and heterogeneous ice nucleation in leaves of tuber-bearing solanum species : ice nucleation activity of external source of nucleants

The heterogeneous ice nucleation characteristics and frost injury in supercooled leaves upon ice formation were studied in nonhardened and cold-hardened species and crosses of tuber-bearing Solanum. The ice nucleation activity of the leaves was low at temperatures just below 0°C and further decreased as a result of cold acclimation. In the absence of supercooling, the nonhardened and cold-hardened leaves tolerated extracellular freezing between −3.5° and −8.5°C. However, if ice initiation in the supercooled leaves occurred at any temperature below −2.6°C, the leaves were lethally injured.

To prevent supercooling in these leaves, various nucleants were tested for their ice nucleating ability. One% aqueous suspensions of fluorophlogopite and acetoacetanilide were found to be effective in ice nucleation of the Solanum leaves above −1°C. They had threshold temperatures of −0.7° and −0.8°C, respectively, for freezing in distilled H₂O. Although freezing could be initiated in the Solanum leaves above −1°C with both the nucleants, 1% aqueous fluorophlogopite suspension showed overall higher ice nucleation activity than acetoacetanilide and was nontoxic to the leaves. The cold-hardened leaves survived between −2.5° and −6.5° using 1% aqueous fluorophlogopite suspension as a nucleant. The killing temperatures in the cold-hardened leaves were similar to those determined using ice as a nucleant. However, in the nonhardened leaves, use of fluorophlogopite as a nucleant resulted in lethal injury at higher temperatures than those estimated using ice as a nucleant.

     

Ice Nucleation Activity in Lichens

A newly discovered form of biological ice nucleus associated with lichens is described. Ice nucleation spectra of a variety of lichens from the southwestern United States were measured by the drop-freezing method. Several epilithic lichen samples of the genera Rhizoplaca, Xanthoparmelia, and Xanthoria had nuclei active at temperatures as warm as −2.3°C and had densities of 2.3 × 10⁶ to more than 1 × 10⁸ nuclei g⁻¹ at −5°C (2 to 4 orders of magnitude higher than any plants infected with ice nucleation-active bacteria). Most lichens tested had nucleation activity above −8°C. Lichen substrates (rocks, plants, and soil) showed negligible activity above −8°C. Ice nucleation-active bacteria were not isolated from the lichens, and activity was not destroyed by heat (70°C) or sonication, indicating that lichen-associated ice nuclei are nonbacterial in origin and differ chemically from previously described biological ice nuclei. An axenic culture of the lichen fungus Rhizoplaca chrysoleuca showed detectable ice nucleation activity at −1.9°C and an ice nucleation density of 4.5 × 10⁶ nuclei g⁻¹ at −5°C. It is hypothesized that these lichens, which are both frost tolerant and dependent on atmospheric moisture, derive benefit in the form of increased moisture deposition as a result of ice nucleation.     

Estimation of the fructose diphosphatase-phosphofructokinase substrate cycle in the flight muscle of Bombus affinis

1. Substrate cycling of fructose 6-phosphate through reactions catalysed by phosphofructokinase and fructose diphosphatase was estimated in bumble-bee (Bombus affinis) flight muscle in vivo. 2. Estimations of substrate cycling of fructose 6-phosphate and of glycolysis were made from the equilibrium value of the ³H/¹⁴C ratio in glucose 6-phosphate as well as the rate of ³H release to water after the metabolism of [5-³H,U-¹⁴C]glucose. 3. In flight, the metabolism of glucose proceeded exclusively through glycolysis (20.4μmol/min per g fresh wt.) and there was no evidence for substrate cycling. 4. In the resting bumble-bee exposed to low temperatures (5°C), the pattern of glucose metabolism in the flight muscle was altered so that substrate cycling was high (10.4μmol/min per g fresh wt.) and glycolysis was decreased (5.8μmol/min per g fresh wt.). 5. The rate of substrate cycling in the resting bumble-bee flight muscle was inversely related to the ambient temperature, since at 27°, 21° and 5°C the rates of substrate cycling were 0, 0.48 and 10.4μmol/min per g fresh wt. respectively. 6. Calcium ions inhibited fructose diphosphatase of the bumble-bee flight muscle at concentrations that were without effect on phosphofructokinase. The inhibition was reversed by the presence of a Ca²⁺-chelating compound. It is proposed that the rate of fructose 6-phosphate substrate cycling could be regulated by changes in the sarcoplasmic Ca²⁺ concentration associated with the contractile process.     

Stabilization of Frozen Lactobacillus delbrueckii subsp. bulgaricus in Glycerol Suspensions: Freezing Kinetics and Storage Temperature Effects

The interactions between freezing kinetics and subsequent storage temperatures and their effects on the biological activity of lactic acid bacteria have not been examined in studies to date. This paper investigates the effects of three freezing protocols and two storage temperatures on the viability and acidification activity of Lactobacillus delbrueckii subsp. bulgaricus CFL1 in the presence of glycerol. Samples were examined at −196°C and −20°C by freeze fracture and freeze substitution electron microscopy. Differential scanning calorimetry was used to measure proportions of ice and glass transition temperatures for each freezing condition tested. Following storage at low temperatures (−196°C and −80°C), the viability and acidification activity of L. delbrueckii subsp. bulgaricus decreased after freezing and were strongly dependent on freezing kinetics. High cooling rates obtained by direct immersion in liquid nitrogen resulted in the minimum loss of acidification activity and viability. The amount of ice formed in the freeze-concentrated matrix was determined by the freezing protocol, but no intracellular ice was observed in cells suspended in glycerol at any cooling rate. For samples stored at −20°C, the maximum loss of viability and acidification activity was observed with rapidly cooled cells. By scanning electron microscopy, these cells were not observed to contain intracellular ice, and they were observed to be plasmolyzed. It is suggested that the cell damage which occurs in rapidly cooled cells during storage at high subzero temperatures is caused by an osmotic imbalance during warming, not the formation of intracellular ice.     

Microbial Life beneath a High Arctic Glacier

The debris-rich basal ice layers of a high Arctic glacier were shown to contain metabolically diverse microbes that could be cultured oligotrophically at low temperatures (0.3 to 4°C). These organisms included aerobic chemoheterotrophs and anaerobic nitrate reducers, sulfate reducers, and methanogens. Colonies purified from subglacial samples at 4°C appeared to be predominantly psychrophilic. Aerobic chemoheterotrophs were metabolically active in unfrozen basal sediments when they were cultured at 0.3°C in the dark (to simulate nearly in situ conditions), producing ¹⁴CO₂ from radiolabeled sodium acetate with minimal organic amendment (≥38 μM C). In contrast, no activity was observed when samples were cultured at subfreezing temperatures (≤−1.8°C) for 66 days. Electron microscopy of thawed basal ice samples revealed various cell morphologies, including dividing cells. This suggests that the subglacial environment beneath a polythermal glacier provides a viable habitat for life and that microbes may be widespread where the basal ice is temperate and water is present at the base of the glacier and where organic carbon from glacially overridden soils is present. Our observations raise the possibility that in situ microbial production of CO₂ and CH₄ beneath ice masses (e.g., the Northern Hemisphere ice sheets) is an important factor in carbon cycling during glacial periods. Moreover, this terrestrial environment may provide a model for viable habitats for life on Mars, since similar conditions may exist or may have existed in the basal sediments beneath the Martian north polar ice cap.     

Fate of Ice Nucleation-Active Pseudomonas syringae Strains in Alpine Soils and Waters and in Synthetic Snow Samples

The stability of the ice nucleation activity (INA) and viability of INA Pseudomonas syringae 31a, used as an ice nucleator in the manufacture of synthetic snow, was determined in snow. The viability of P. syringae 1-2b, a rifampin-resistant mutant selected from strain 31a to improve recovery from test samples, was determined in laboratory tests of three alpine soil and water samples from three different sources. Snow samples were exposed to environmental conditions or held in darkness at −20°C. Samples of soil and water were maintained in darkness at 0, 7.5, or 15°C. Parent strain 31a INA decreased significantly (>99.0%) in snow exposed to sunlight and freeze-thaw, while the INA of the cell population in snow held in darkness at −20°C remained essentially unchanged. No viable strain 31a was detected in snow exposed to the environment after 7 days, while the viability of strain 31a in snow held in darkness at −20°C decreased to <3% of the original inoculation at the test conclusion. Mutant strain 1-2b viability was undetectable or had decreased significantly 19 days postinoculation in soil samples held at 0 or 15°C. In contrast, 1-2b viability remained detectable at low levels for the duration of the test in soils held at 7.5°C. The 1-2b population demonstrated a significantly longer half-life in peatlike soil than in the loam soils tested. The rate of decrease in 1-2b viability was essentially the same in the three alpine water samples tested with respect to water temperature and sample location.     

Effects of Incubation Temperature on Growth and Production of Exopolysaccharides by an Antarctic Sea Ice Bacterium Grown in Batch Culture

The sea ice microbial community plays a key role in the productivity of the Southern Ocean. Exopolysaccharide (EPS) is a major component of the exopolymer secreted by many marine bacteria to enhance survival and is abundant in sea ice brine channels, but little is known about its function there. This study investigated the effects of temperature on EPS production in batch culture by CAM025, a marine bacterium isolated from sea ice sampled from the Southern Ocean. Previous studies have shown that CAM025 is a member of the genus Pseudoalteromonas and therefore belongs to a group found to be abundant in sea ice by culture-dependent and -independent techniques. Batch cultures were grown at −2°C, 10°C, and 20°C, and cell number, optical density, pH, glucose concentration, and viscosity were monitored. The yield of EPS at −2°C and 10°C was 30 times higher than at 20°C, which is the optimum growth temperature for many psychrotolerant strains. EPS may have a cryoprotective role in brine channels of sea ice, where extremes of high salinity and low temperature impose pressures on microbial growth and survival. The EPS produced at −2°C and 10°C had a higher uronic acid content than that produced at 20°C. The availability of iron as a trace metal is of critical importance in the Southern Ocean, where it is known to limit primary production. EPS from strain CAM025 is polyanionic and may bind dissolved cations such at trace metals, and therefore the presence of bacterial EPS in the Antarctic marine environment may have important ecological implications.     

Studies on the effects of subzero temperatures on the viability of spores of Aspergillus flavus. I. The effect of rate of warming

1. The survival of spores of Aspergillus flavus suspended in distilled water and cooled rapidly to –70 to –75°C. was found to depend primarily on the rate of subsequent warming of the frozen suspension. Only 7 per cent of the spores germinated following slow warming at 0.9°C. per minute, whereas about 75 per cent germinated following rapid warming at 700°C. per minute. 2. Viability was dependent on the rate at which the suspensions warmed from –70 to 0°C. (subzero warming), but was not dependent on the rate of thawing of the frozen water in which the spores were suspended. 3. The logarithm of the percentage of germination appeared to be a linear function of the logarithm of the rate of subzero warming when spores were warmed at rates ranging from 0.12 to 1000°C. per minute. 4. The lethal effects of slow warming from –70 to 0°C. were more pronounced between about –20 and 0°C. than between –70 and –20°C. In the former range of temperatures, the percentage of germination decreased sharply as slow warming progressed towards 0°C. 5. Slow warming from –70 to 0°C. was more harmful to the spores than was a 1 or 2 hour exposure to constant temperatures between –70 and 0°C. 6. Slow warming was found to be more harmful than rapid warming when spores were suspended in horse serum, 0.16 molal sodium chloride, or 0.29 molal sucrose as well as in distilled water.     

Relaxed tarantula skeletal muscle has two ATP energy-saving mechanisms

Myosin molecules in the relaxed thick filaments of striated muscle have a helical arrangement in which the heads of each molecule interact with each other, forming the interacting-heads motif (IHM). In relaxed mammalian skeletal muscle, this helical ordering occurs only at temperatures >20°C and is disrupted when temperature is decreased. Recent x-ray diffraction studies of live tarantula skeletal muscle have suggested that the two myosin heads of the IHM (blocked heads [BHs] and free heads [FHs]) have very different roles and dynamics during contraction. Here, we explore temperature-induced changes in the BHs and FHs in relaxed tarantula skeletal muscle. We find a change with decreasing temperature that is similar to that in mammals, while increasing temperature induces a different behavior in the heads. At 22.5°C, the BHs and FHs containing ADP.P_i are fully helically organized, but they become progressively disordered as temperature is lowered or raised. Our interpretation suggests that at low temperature, while the BHs remain ordered the FHs become disordered due to transition of the heads to a straight conformation containing Mg.ATP. Above 27.5°C, the nucleotide remains as ADP.P_i, but while BHs remain ordered, half of the FHs become progressively disordered, released semipermanently at a midway distance to the thin filaments while the remaining FHs are docked as swaying heads. We propose a thermosensing mechanism for tarantula skeletal muscle to explain these changes. Our results suggest that tarantula skeletal muscle thick filaments, in addition to having a superrelaxation–based ATP energy-saving mechanism in the range of 8.5–40°C, also exhibit energy saving at lower temperatures (<22.5°C), similar to the proposed refractory state in mammals.     

Juxtaposition of the changes in intracellular calcium and force during staircase potentiation at 30 and 37°C

Ca²⁺ entry during the action potential stimulates muscle contraction. During repetitive low frequency stimulation, skeletal muscle undergoes staircase potentiation (SP), a progressive increase in the peak twitch force induced by each successive stimulus. Multiple mechanisms, including myosin regulatory light chain phosphorylation, likely contribute to SP, a temperature-dependent process. Here, we used the Ca²⁺-sensitive fluorescence indicators acetoxymethyl (AM)-furaptra and AM-fura-2 to examine the intracellular Ca²⁺ transient (ICT) and the baseline Ca²⁺ level at the onset of each ICT during SP at 30 and 37°C in mouse lumbrical muscle. The stimulation protocol, 8 Hz for 8 s, resulted in a 27 ± 3% increase in twitch force at 37°C and a 7 ± 2% decrease in twitch force at 30°C (P < 0.05). Regardless of temperature, the peak rate of force production (+df/dt) was higher in all twitches relative to the first twitch (P < 0.05). Consistent with the differential effects of stimulation on twitch force at the two temperatures, raw ICT amplitude decreased during repetitive stimulation at 30°C (P < 0.05) but not at 37°C. Cytosolic Ca²⁺ accumulated during SP such that baseline Ca²⁺ at the onset of ICTs occurring late in the train was higher (P < 0.05) than that of those occurring early in the train. ICT duration increased progressively at both temperatures. This effect was not entirely proportional to the changes in twitch duration, as twitch duration characteristically decreased before increasing late in the protocol. This is the first study identifying a changing ICT as an important, and temperature-sensitive, modulator of muscle force during repetitive stimulation. Moreover, we extend previous observations by demonstrating that contraction-induced increases in baseline Ca²⁺ coincide with greater +df/dt but not necessarily with higher twitch force.     

Effect of low temperature and calcium on survival and membrane properties of isolated winter wheat cells

Isolated cells obtained by enzymic digestion of young primary leaves of cold-hardened, dark-grown Kharkov winter wheat (Triticum aestivum L.) were exposed to various low temperature stresses. The initial uptake of ⁸⁶Rb was generally decreased by increasing concentrations of Ca²⁺, but after longer periods of incubation, the inhibiting effect of high Ca²⁺ levels diminished. Viability of isolated cells suspended in water declined rapidly when ice encased at −1°C, while in the presence of 10 millimolar Ca²⁺ viability declined only gradually over a 5-week period. Ice encasement markedly reduced ⁸⁶Rb uptake prior to a significant decline in cell viability or increased ion efflux. Cell damage increased progressively when the icing temperature was reduced from −1 to −2 and −3°C, but the presence of Ca²⁺ in the suspending medium reduced injury. Cell viability and ion uptake were reduced to a greater extent following slow cooling than after rapid cooling to subfreezing temperatures ranging from −10 to −30°C. The results from this study support the view that an early change in cellular properties due to prolonged ice encasement at −1°C involves the ion transport system, whereas cooling to lower subfreezing temperatures for only a few hours results in more general membrane damage, including loss of semipermeability of the plasma membrane.