Kinetics of water loss and the likelihood of intracellular freezing in mouse ova. Influence of the method of calculating the temperature dependence of water permeability

To avoid intracellular freezing and its usually lethal consequences, cells must lose their freezable water before reaching their ice-nucleation temperature. One major factor determining the rate of water loss is the temperature dependence of the water permeability, Lp (hydraulic conductivity). Because of the paucity of water permeability measurements at subzero temperatures, that temperature dependence has usually been extrapolated from above-zero measurements. The extrapolation has often been based on an exponential dependence of Lp on temperature. This paper compares the kinetics of water loss based on that extrapolation with that based on an Arrhenius relation between Lp and temperature, and finds substantial differences below -20 to -25 degrees C. Since the ice-nucleation temperature of mouse ova in the cryoprotectants DMSO and glycerol is usually below -30 degrees C, the Arrhenius form of the water-loss equation was used to compute the extent of supercooling in ova cooled at rates between 1 and 8 degrees C/min and the consequent likelihood of intracellular freezing. The predicted likelihood agrees well with that previously observed. The water-loss equation was also used to compute the volumes of ova as a function of cooling rate and temperature. The computed cell volumes agree qualitatively with previously observed volumes, but differ quantitatively.     

Thermodynamic analysis of the physical state of water during freezing in plant tissue, based on the temperature dependence of proton spin-spin relaxation

Multi-proton spin-echo images were collected from cold-acclimated winter wheat crowns (Triticum aestivum L.) cv. Cappelle Desprez at 400 MHz between 4 and ?4 °C. Water proton relaxation by the spin-spin (T2) mechanism from individual voxels in image slices was found to be mono-exponential. The temperature dependence of these relaxation rates was found to obey Arrhenius or absolute rate theory expressions relating temperature, activation energies and relaxation rates, Images whose contrast is proportional to the Arrhenius activation energy (E_a), Gibb's free energy of activation (ΔG^?), and the entropy of activation (ΔS^?) for water relaxation on a voxel basis were constructed by post-image processing. These new images exhibit contrast based on activation energies rather than rules of proton relaxation. The temperature dependence of water proton T₂ relaxation rates permits prediction of changes in the physical state of water in this tissue over modest temperature ranges. A simple model is proposed to predict the freezing temperature kof various tissue in wheat crowns. The average E_a and ΔH^? for water proton T₂ relaxation over the above temperature range in winter wheat tissue were ?6.4 ± 14.8 and ?8.6 ± 14.8kj mol^?1, respectively. This barrier is considerably lower than the E_a for proton translation in ice at 0°C, which is reported to be between 46.0 and 56.5 kj mol^?1     

Permeability of isolated rat hepatocyte plasma membranes for molecules of dimethyl sulfoxide

We have studied permeability of isolated rat hepatocyte membranes for molecules of dimethyl sulfoxide (DMSO) at different hypertonicity of a cryoprotective medium. The permeability coefficient of hepatocyte membranes k ₁ for DMSO molecules was shown to be the differential function of osmotic pressure between a cell and an extracellular medium. Ten-fold augmentation of DMSO concentration in the cryoprotective medium causes the decrease of permeability coefficients k ₁ probably associated with the increased viscosity in membrane-adjacent liquid layers as well as partial limitations appeared as a result of change in cell membrane shape after hepatocyte dehydration. We have found out that in aqueous solutions of NaCl (2246 mOsm/L) and DMSO (2250 mOsm/L) the filtration coefficient L _p in the presence of a penetrating cryoprotectant (L _pDMSO = (4.45 ± 0.04) · 10^?14 m³/Ns) is 3 orders lower compared to the case with electrolyte (L _pNaCl = (2.25 ± 0.25) · 10^?11 m³/Ns). This phenomenon is stipulated by the cross impact of flows of a cryoprotectant and water at the stage of cell dehydration. Pronounced lipophilicity of DMSO, geometric parameters of its molecule as well as the presence of large aqueous pores in rat hepatocyte membranes allow of suggesting the availability of two ways of penetrating this cryoprotectant into the cells by non-specific diffusion through membrane lipid areas and hydrophilic channels.     

Age and growth of Siberian sculpin (Cottus poecilopus) and young brown trout (Salmo trutta) in a subalpine Norwegian river

Age, growth and density of Siberian sculpin (Cottus poecilopus) and young brown trout (Salmo trutta) within two sections of River Atna; above Lake Atnsjøen [Section 1 at altitudes between 739 and 715 m] and below Lake Atnsjøen [Section 2 at altitudes between 430 and 370 m] was studied during a 6-year period (1986–91). The water temperature was considerably lower in Section 1 than in Section 2, as the number of days with a water temperature above 10?°C (T _{D ≥ 10?° C} ) from spring to August 1 ranged between 2–26 and 26–52 days, respectively. Juvenile brown trout (age 0+) attained a significantly smaller body size in Section 1 than in Section 2; mean length ±SD was 35 ± 8 mm (ranged 27–46) and 43 ± 7 mm (range 38–46), respectively. In Section 2, there was a highly positive correlation between the body length of 0+ brown trout and mean water temperature in June (p<0.005), and also to some extent in Section 1 (p=0.11). Individuals of age 1+ did not exhibit any such difference, while fish in age group 2+ were larger in Section 1 than in Section 2. By using the number of days with a water temperature between the range 5–10?°C (T _{D ≥ 5 ? 10?° C} ) as test variables, we found a highly positive correlation between the August 1 body length of 0+ brown trout and T _{D ≥ 9}?°C from spring to August 1 in Section 2 (p<0.05), as opposed to T _{D ≥ 7}?°C for trout in Section 1 (p=0.11). Young Siberian sculpin (age 0+ and 1+) also exhibited slower growth in Section 1 than in Section 2, but this was not the case among older specimens. In the year with the lowest temperature measured (1987), no 0+ Siberian sculpin were caught in any of the two sections, indicating that low temperature affects their survival. Both species exhibited large spatial and temporal variation in density. Thus, data on abundance and growth sampled on one occasion at one site can not be regarded as representative for these two fish populations.     

Cooperativity in viral fusion

A simple model for membrane fusion mediated by vial spike glycoproteins is presented. The viral proteins are considered to be allosteric proteins that undergo concerted conformational transitions when they bind the ligand. The ligand in this case is H⁺. The effect of the conformational transition is to bring membranes together and induce their fusion. An equation is derived for the dependence of fusion rates on ligand concentration, for a given dissociation constant (K _d), equilibrium constant for the conformational change (L), and number of cooperating subunits (n). Curves generated by this equation provide a reasonable fit to data on the rates of fusion of Vesicular Stomatitis virus with cells for a pK _d of 6.3,L=1000 andn=6.     

Purification and properties of acetate kinase from Clostridium thermoaceticum

Acetate kinase (ATP: acetate phosphotransferase EC 2.7.2.1) has been purified from Clostridium thermoaceticum. The enzyme of a specific activity of 282 μmoles min^-1 mg^-1 appeared homogeneous as judged from Sephadex chromatography and sedimentation velocity. Polyacrylamide gel electrophoretic patterns at pH 9.0 and 9.5 showed heterogeneity. Velocity curves obtained with varying amount of acetate were of the Michaelis-Menten type with an apparent K _m of 0.135 M. With varying amounts of ATP sigmoidal kinetic was observed (S_0.5=1.64 mM), suggesting cooperative binding of this substrate. The enzyme had only moderate thermal stability with a temperature optimum of about 60°C and exhibited a broken line in an Arrhenius graph. From gel filtration a molecular weight of about 60 000 daltons was estimated for the enzyme. The S_20w value was 6.0 S.     

Hydraulic adjustments in aspen (Populus tremuloides) seedlings following defoliation involve root and leaf aquaporins

Main conclusion

Changes in root and leaf hydraulic properties and stimulation of transpiration rates that were initially triggered by defoliation were accompanied by corresponding changes in leaf and root aquaporin expression. Aspen (Populus tremuloides) seedlings were subjected to defoliation treatments by removing 50, 75 % or all of the leaves. Root hydraulic conductivity (L_pr) was sharply reduced in plants defoliated for 1 day and 1 week. The decrease in L _pr could not be prevented by stem girdling and it was accompanied in one-day-defoliated plants by a large decrease in the root expression of PIP1,2 aquaporin and an over twofold decrease in hydraulic conductivity of root cortical cells (L _pc). Contrary to L _pr and L _pc, 50 and 75 % defoliation treatments profoundly increased leaf lamina conductance (K _lam) after 1 day and this increase was similar in magnitude for both defoliation treatments. Transpiration rates (E) rapidly declined after the removal of 75 % of leaves. However, E increased by over twofold in defoliated plants after 1 day and the increases in E and K _lam were accompanied by five- and tenfold increases in the leaf expression of PIP2;4 in 50 and 75 % defoliation treatments, respectively. Defoliation treatments also stimulated net photosynthesis after 1 day and 3 weeks, although the increase was not as high as E. Leaf water potentials remained relatively stable following defoliation with the exception of a small decrease 1 day after defoliation which suggests that root water transport did not initially keep pace with the increased transpirational water loss. The results demonstrate the importance of root and leaf hydraulic properties in plant responses to defoliation and point to the involvement of PIP aquaporins in the early events following the loss of leaves.     

Interactive effects of near-future temperature increase and ocean acidification on physiology and gonad development in adult Pacific sea urchin,Echinometra sp. A

Increased atmospheric CO₂ will have a twofold impact on future marine ecosystems, increasing global sea surface temperatures and uptake of CO₂ (Ocean Acidification). Many experiments focus on the investigation of one of these stressors, but under realistic future climate predictions, these stressors may have interactive effects on individuals. Here, we investigate the effect of warming and acidification in combination. We test for interactive effects of potential near-future (2100) temperature (+2 to 3 °C) and pCO₂ (~860–940 μAtm) levels on the physiology of the tropical echinoid Echinometra sp. A. The greatest reduction in growth was under simultaneous temperature and pH/pCO₂ stress (marginally significant temperature × pH/pCO₂ interaction). This was mirrored by the physiological data, with highest metabolic activity (measured as respiration and ammonium excretion) occurring at the increased temperature and pCO₂ treatment, although this was not significant for excretion. The perivisceral coelomic fluid pH was ~7.5–7.6, as typical for echinoids, and showed no significant changes between treatments. Indicative of active calcification, internal magnesium and calcium concentrations were reduced compared to the external medium, but were not different between treatments. Gonad weight was lower at the higher temperature, and this difference was more distinct and statistically significant for males. The condition of the gonads assessed by histology declined in increased temperature and low pH treatments. The Echinometra grew in all treatments indicating active calcification of their magnesium calcite tests even as carbonate mineral saturation decreased. Our results indicate that the interactive temperature and pH effects are more important for adult echinoids than individual stressors. Although adult specimens grow and survive in near-future conditions, higher energy demands may influence gonad development and thus population maintenance.     

Effects of Mild and Moderate Hypothemia Therapy on Expression of Cerebral Neuron Apoptosis Related Proteins and Glial Fiber Acidic Protein After Rat Cardio-pulmonary Resuscitation

To explore the effects of different degrees of hypothermia on brain tissue apoptosis after cardio-pulmonary resuscitation (CPR). Cardiac arrest for 5 min induced by asphyxia method was used to create CPR model. 30 SD rats were randomly divided into control group (normothermia), 33 °C hypothermia group and 30 °C hypothermia group with ten rats in each. Rats in control group received routine treatment at 25 °C room temperature after CPR; Rats in mild hypothermia and moderate hypothermia groups were given hypothermia treatment 0.5 h after CPR. Brain tissue in all groups was taken 24 h after CPR, and immunohistochemistry was used to detect the caspase-3 in cerebral cortex and glial fiber acidic protein (GFAP) expression in astrocyte. Western blotting was used to detect Bcl-2 and Bax protein expression, and histopathological change was observed in brain tissue. Compare to the control group, caspase-3 expression in cerebral neurons in hypothermia group was significantly decreased (p<0.01), which was significantly lower in 30 °C group than that in 33 °C group (p > 0.05); GFAP level in hypothermia groups was significantly increased (p < 0.01), which was higher in 30 °C hypothermia group than that in 33 °C hypothermia group (p < 0.05); Bcl-2 expression level in hypothermia group was significantly increased (p < 0.01), which was higher in 30 °C hypothermia group than that in 33 °C hypothermia group (p < 0.05); The level of Bax had no significant difference among the three groups. Hypothermia-regulated GFAP expression by decreasing caspase-3 expression and increasing Bcl-2 expression to promote brain cell signaling transduction, and further inhibited cell apoptosis and reduced brain injury. Moderate hypothermia therapy is more effective than mild hypothermia in preventing brain injure.     

The kinetics of light-induced changes of C-550, cytochrome b559 and fluorescence yield in chloroplasts at low temperature

The kinetics of the photoreduction of C-550, the photooxidation of cytochrome b₅₅₉ and the fluorescence yield changes during irradiation of chloroplasts at ?196 °C were measured and compared. The photoreduction of C-550 proceeded more rapidly than the photooxidation of cytochrome b₅₅₉ and the fluorescence yield increase followed the cytochrome b₅₅₉ oxidation. These results suggest that fluorescence yield under these conditions indicates the dark reduction of the primary electron donor to Photosystem II, P680⁺, by cytochrome b₅₅₉ rather than the photoreduction of the primary electron acceptor.The photoreduction of C-550 showed little if any temperature dependence over the range of ?196 to ?100 °C. The amount of cytochrome b₅₅₉ photooxidized was sensitive to temperature decreasing from the maximal change at temperatures between ?196 to ?160 °C to no change at ?100 °C. To the extent that the reaction occurred at temperatures between ?160 and ?100 °C the rate was largely independent of temperature. The rate of the fluorescence increase was dependent on temperature over this range being 3–4 times more rapid at ?100 than at ?160 °C. At ?100 °C the light-induced fluorescence increase and the photoreduction of C-550 show similar kinetics. The temperature dependence of the fluorescence induction curve is attributed to the temperature dependence of the dark reduction of P680⁺.The intensity dependence of the photoreduction of C-550 and of the photooxidation of cytochrome b₅₅₉ are linear at low intensities (below 200 μW/cm²) but fall off at higher intensities. The failure of reciprocity in the photoreduction of C-550 at the higher intensities is not explained by the simple model proposed for the Photosystem II reaction centers.     

Red blood cell membrane water permeability increases with length of ex vivo storage

Water transport across the red blood cell (RBC) membrane is an essential cell function that needs to be preserved during ex vivo storage. Progressive biochemical depletion during storage can result in significant conformational and compositional changes to the membrane. Characterizing the changes to RBC water permeability can help in evaluating the quality of stored blood products and aid in the development of improved methods for the cryopreservation of red blood cells. This study aimed to characterize the water permeability (L_p), osmotically inactive fraction (b), and Arrhenius activation energy (E_a) at defined storage time-points throughout storage and to correlate the observed results with other in vitro RBC quality parameters. RBCs were collected from age- and sex-matched blood donors. A stopped flow spectrophotometer was used to determine L_p and b by monitoring changes in hemoglobin autofluorescence when RBCs were exposed to anisotonic solutions. Experimental values of L_p were characterized at three different temperatures (4, 20 and 37 °C) to determine the E_a. Results showed that L_p, b, and E_a of stored RBCs significantly increase by day 21 of storage. Degradation of the RBC membrane with length of storage was seen as an increase in hemolysis and supernatant potassium, and a decrease in deformability, mean corpuscular hemoglobin concentration and supernatant sodium. RBC osmotic characteristics were shown to change with storage and correlate with changes in RBC membrane quality metrics. Monitoring water parameters is a predictor of membrane damage and loss of membrane integrity in ex vivo stored RBCs.     

The erythrocyte membrane in essential hypertension characterization of the temperature dependence of lithium efflux

Erythrocytes of most patients with essential hypertension are distinguished by a typical pattern of temperature-dependence of Li efflux. In the present study we have attempted to characterize this unique temperature response. Measurements of Li efflux into Na medium and Li_i-Na_o countertransport were conducted simultaneously at finely spaced temperature intervals with increments of 1 to 2°C in the range of 10–40°C. The Arrhenius plots for the efflux in Na medium and for Li_i-Na_o countertransport in erythrocytes of both normotensives and hypertensives were biphasic with slopes representing apparent energies of activation of about 28 and 8 kcal/mol below and above the ‘break’, respectively. However, the ‘break’ in the Arrhenius plot appeared at distinctly different temperatures: 30°C for normotensives and 20°C for hypertensives. The Li efflux was resolved into N-ethylmaleimide-sensitive and -insensitive components. The sensitive component exhibited a typical biphasic temperature response, with the characteristic ‘break’: at 30°C for normotensives and at 20°C for hypertensives. In contrast, the N-ethylmaleimide-insensitive component was alike in normotensives and hypertensives. It is concluded that: (a) the unique temperature dependence of Li efflux in erythrocytes of hypertensives results from a localized modification in the membrane; (b) the N-ethylmaleimide-sensitive component represents a protein moiety which distinguishes between the erythrocyte membrane of normotensives and hypertensives; (c) the expression of the temperature dependence as judged by the sharp transition in slope (within 1 to 2°C), apparently reflects the cooperative involvement of membrane lipids, associated with the Li efflux system.     

Comparative thermodynamics of opioid receptor ligand interaction in the bovine adrenal medulla membranes—Evidence of opioid site heterogeneity

1. A marked dependence on temperature of agonist binding δ, μ and κ₁₋₃, opioid sites in the bovine adrenal medulla was observed, at the range of 0 to 37°C. These changes concern kinetic (k₁) and equilibrium constants (K_d), but not binding capacities (B_max).2. These dependences are different for each ligand and each opioid receptor, suggesting their molecular heterogeneity.3. The comparative thermodynamics indicates that the interaction of opioid agonists with their receptor is exergonic (ΔG° < 0) and entropy driven (ΔS° > 0).4. The comparison of Van't Hoff and Arrhenius plots indicates a discrete mechanism in the binding of each opioid receptor.     

Affinity labeling of the active site of pig liver NADH-cytochrome b5 reductase by 5′-p-fluorosulfonylbenzoyladenosine

Lysosome-solubilized pig liver NADH-cytochrome b₅ reductase is inactivated by 5′-p-fluorosulfonylbenzoyladenosine (5′-FSBA) following pseudo-first-order kinetics. A double reciprocal plot of 1/K _obs versus 1/[5′-FSBA] yields a straight line with a positiveY intercept, indicative of reversible binding of the analogue prior to an irreversible incorporation.K _d or the initial reversible enzyme-analogue complex is estimated at 185 µM withK ₂=0.22 min^?1 (atpH 8.0 and 25°C). A stoichiometry of 1.2 moles of analogue bound/mole of enzyme at 100% inactivation has been determined from incorporation studies using 5′-p-fluorosulfonylbenzoyl-[¹⁴C]adenosine. The irreversible inactivation as well as the covalent incorporation could be completely prevented by the presence of NADH, the substrate of enzyme, during the incubation. Four 5′-FSBA-labeled peptides were isolated by reverse-phase high-performance liquid chromatography of tryptic digest of the modified NADH-cytochrome b₅ reductase and their amino acid sequences were determined. These peptides appear to be related to the NADH binding site of the enzyme.     

The effects of temperature and light intensity on growth,reproduction and EPS synthesis of a thermophilic strain related to the genus Graesiella

Tunisian microalgae are diverse and rarely been studied. This study reports a first investigation of thermophile Chlorophyta isolated from mats community colonizing the geothermal springs in the north of Tunisia at water temperature 60 °C. In the study, the combined effect of temperature and light intensity was investigated on the cell growth, the mother and daughter cells abundance and the extracellular polymeric substances synthesis in batch culture of the isolated species. Three levels were tested for each factor, 20, 30, 40 °C for temperature; and 20, 70, 120 μmol photons m^?2 s^?1 for light intensity, using full factorial design and response surface methodology. The thermophile strain was identified as a genus Graesiella and showed 99.8 % similarity with two Graesiella species: Graesiella emersonii and Graesiella vacuolata based on the 18S rDNA molecular identification. The optimal growth condition was found at 30 °C and 120 µmol photons m^?2 s^?1 (7 MC mL^?1 day^?1), with the abundance of vegetative cells (daughter cells). In contrast, the number of mother cells increased significantly as the growth decreased; consequently, the highest ratio of auto spore mother cells versus daughter cells (19.4) was obtained at 20 °C and 20 µmol photons m^?2 s^?1. The highest yield of EPS production (11.7 mg L^?1 day^?1) was recorded at the highest temperature (40 °C) and lowest light intensity (20 µmol photons m^?2s^?1). These results revealed how the species respond to high and low temperatures and suggest that the species should be considered as facultative thermophile.     

Evidence for a glucosyl-enzyme intermediate in the beta-glucosidase-catalyzed hydrolysis of p-nitrophenyl-beta-D-glucoside

The reaction of almond β-glucosidase with $\text{p}$-nitrophenyl-β-$\text{D}$-glucoside has been investigated over the temperature range +25° to ?45° using 50% aqueous dimethyl sulfoxide (DMSO) as solvent. At temperatures below those at which turnover occurs a “burst” of $\text{p}$-nitrophenol proportional to the enzyme concentration is observed. Such a “burst” suggests the existence of a glucosyl-enzyme intermediate whose breakdown is rate-limiting, and provides a method for measuring the active-site normality. At pH 5.9, 25°, the presence of 50% DMSO causes an increase in K_m from 1.7×10^?3M (0%) to 1.7×10^?2M, whereas V_max is unchanged. The DMSO thus apparently acts as a competitive inhibitor with K_i = 0.7M. The Arrhenius plot for turnover is linear over the accessible temperature range with E_a = 23.0 ± 2.0 kcal/mole.     

Membrane phase transitions and succinate oxidase activity in an extremely thermophilic bacterium

ESR and succinate oxidase activity were used to investigate the membrane phase-transitions of an extreme thermophile, Thermus T351, over an 80°C temperature range in whole cells, membrane particles, and extracted lipid suspension. Three phase transitions were observed using both techniques. These occurred at about 19°C, 39°C and 66°C. The transition at 19°C is unusual in that the Arrhenius plot for succinate oxidase is concave upwards, implying an increase in activation energy (E_a) with increased temperature.