Requirement of heat and metabolic energy for the expression of inhibitory action of colicin K.

Escherichia coli B, induced for beta-galactoside permease, can accumulate thio-methyl-beta-galactoside in the cell even at 0 degrees D. At this temperature, cells adsorb colicin K but the adsorbed colicin does not inhibit thiomethyl-beta-galactoside uptake. Inhibition by colicin K is, however, seen at 0 degrees C after exposure of the colicin K-cell complex to a high temperature: a greater degree of inhibition occurs with increasing temperature or duration or exposure. There is a transition point at around 21 degrees C in Arrhenius plots of this colicin K activation reaction. If inhibitors of energy yielding reactions are present during the heat treatment, the inhibitory action of colicin K (as measured by thiomethyl-beta-galactoside uptake after returning the colicin K-cell complex to 0 degrees C and removal of the inhibitors) is prevented. These results indicate that adsorbed colicin K is converted into the active state only in the presence of metabolic energy and that cell surface fluidity appears to be concerned in this process.     

Change of thermal stability of colicin E7 triggered by acidic pH suggests the existence of unfolded intermediate during the membrane-translocation phase

Purified colicin E7 was analyzed by CD spectrum and gel filtration chromatography in a mimicking membrane-translocation phase. It was found that the CD spectra of colicin E7 at pH 7 and pH 2.5 were similar. Although the melting temperature of the protein shifted from 54.5°C to 34°C at low pH, the thermal denaturation curves of colicin E7 at different pH conditions still fit a two-state model. These experimental results imply that a minor structural change, triggered by acidic pH, for instance, may reduce the energy required for protein melting. In contrast to the minor change in secondary structure at different pH conditions, we observed that, in vitro, all monomeric colicin E7s converted into multimer-like conformations after recovering from the partial unfolding process. This multimeric form of colicin can only be dissociated by formamide and guanidine hydrochloride, indicating that this protein complex is indeed formed by aggregation of the monomeric colicins. Most interestingly, the aggregated colicins still perform in vivo bacteriocidal activity. We suggest that in a partial unfolding state the colicin is prepared for binding to the specific targets for translocation through the membrane. However, in the absence of specific targets in vitro these unfold intermediates may therefore aggregate into the multimeric form of colicins. Proteins 32:17–25, 1998. © 1998 Wiley-Liss, Inc.     

Heat-induced stimulation of 3-O-methylglucose transport in rat thymocytes.

Cells incubated at 41–46 °C show a gradual increase in the initial rate of 3-O-methylglucose uptake when subsequently assayed at 37 °C. Cellular ATP levels remain constant throughout this temperature range, but at temperatures higher than 46 °C, ATP levels decline as does the extent of transport stimulation. Cells incubated at 45 °C for 5 min continue to show a gradual increase in transport activity throughout a subsequent 25-min incubation period at 37 °C. The increase in transport activity is characterized by an increase in the proportion of the rapid phase of 3-O-methylglucose uptake, with little or no change in the half-time of either the rapid phase or the slow phase. Transport stimulation at high temperatures is blocked by inhibitors of oxidative phosphorylation. Cells depleted of intracellular exchangeable Ca²⁺ by treatment with the ionophore A23187 in the presence of ethylene glycol bis(β-aminoethyl ether)-N,N′-tetraacetic acid show nearly the same degree of stimulation at high temperatures as untreated cells, suggesting that exchangeable Ca²⁺ ions do not play an obligatory role in the mechanism of transport stimulation. It is suggested that structural changes occur at 41–46 °C in the membrane proteins controlling glucose transport activity.     

Characteristics of ion transport in kidney cortex of mammalian hibernators

Slices of kidney cortex of two species of hibernating mammals (hamsters and ground squirrels) have been leached of K, and their subsequent ability to reaccumulate K in vitro has been determined at temperatures between 38° and 0°C. At 5°C (body temperature of a hibernating mammal) uptake is appreciable in kidney cortex of both species. In the kidney cortex of hamsters, for example, the tissue K of slices incubated at 5°C reaches the same steady-state concentration after 2 hours that is observed in slices at 38°C after 20 minutes. At 0°C there is also a measurable uptake. This K transport is blocked by metabolic inhibitors and, in ground squirrel kidneys, by ouabain. In kidney cortex slices from guinea pigs net K accumulation is slight at 5°C and absent at 0°C. The initial rapid uptake of K at 38°C occurs at the same rate in kidney cortex slices of hamsters as in those of rabbits. Lowering the temperature of incubation decreases this initial rate of uptake in hamster kidney slices with a Q ₁₀ of 1.8 between 38° and 15° and of 5.7 between 15° and 0°C. In hamsters this uptake of K has been shown to require the outward extrusion of Na. Conversely, about half of the outward extrusion of Na requires K in the medium, while the remainder appears to be independent of K. The conclusions warranted are that kidney cells of hibernators possess an unusual ability to transport ions at low temperature, that this ability does not depend upon a more rapid rate at higher temperatures, and that the characteristics of transport at low temperature are qualitatively similar to those at 38°C in cells of nonhibernators.     

Autolysis of Bacillus subtilis induced by low temperature

By exposure to a temperature below the membrane phase transition point, Bacillus subtilis 168 lost their permeability control followed by the leakage of intracellular K⁺ and incapability of glucose uptake, resulting in cellular lysis in the following incubation at 37°C in the presence of a high concentration (∼ 100 mM) of monovalent cations. The result confirmed that the concomitance of energy deprivation and the presence of monovalent cations were the factors that caused the lysis observed after low temperature exposure.     

Effects of ammonium chloride and chloroquine on endocytic uptake of liposomes by Kupffer cells in vitro

In this study we investigated the interaction of liposomes with rat Kupffer cells in maintenance culture by using the lysosomotropic amines ammonium chloride and chloroquine as inhibitors of intralysosomal degradation. The liposomes (large unilamellar vesicles) contained either the metabolically inert ³H-labeled inulin or the degradable ¹²⁵I-labeled bovine serum albumin. In control incubations, the cells released nearly all accumulated protein label and about 30% of the lipid label when they were incubated in the absence of liposomes, after an initial uptake period of 1 h in the presence of liposomes. This release of label was, for the greater part, suppressed in the presence of ammonia or chloroquine. When the inhibitors were present during the initial uptake period, a several-fold increase in the amount of protein label accumulating in the cells and a smaller, but still marked, increase in lipid label accumulation were observed. The effect of ammonia when present during uptake was readily reversible in contrast to that of chloroquine. Experiments with encapsulated inulin revealed that both lysosomotropic agents also affected the uptake process per se to some extent, probably as a result of impaired membrane/receptor recycling. Labeled liposomes adsorbed to the cells at 4°C were effectively internalized and processed intracellulary after shifting the temperature to 37°C, even when a 500-fold excess of unlabeled liposomes was present in the medium during the 37°C incubation. The observed effects of ammonia and chloroquine indicate that, after uptake, the liposomes are degraded within lysosomes, thus confirming our previous conclusion that endocytosis is the major uptake mechanism at 37°C. From the temperature-change experiments we conclude that, at 4°C, the liposomes are bound with high affinity to the cells, remaining firmly attached to the cell-surface structures which initiate their internalization when the temperature is raised to 37°C.     

The effect of temperature on the uptake and loss of anions by seedling roots ofZea mays L

Intact maize seedlings were examined for the uptake and leakage of labelled sulphate and phosphate anions affected by temperature. Control plants, grown at 25 °C were compared from the aspects of uptake capacity and leakage with plants incubated in nutrient solutions cooled to 15 °C and 5 °C, respectively. Short time intervals as well as 1–7 d exposure to cooling were used. Already after 1 h exposure at 5 °C and 5 h cooling at 15 °C and at 5 °C, considerable changes were manifested in anion uptake and leakage. The uptake of³²P declined more than that of³⁵S. So, after a 30 min uptake interval the uptake of³⁵S decreased at 15 °C to 49.84% and at 5 °C to 6.05% comparing with the uptake at 25 °C, while the uptake of³²P declined to 28.64% at 15 °C and to 4.45% at 5 °C. The leakage of both anions was the highest at 25 °C in absolute rates, but relatively most of the uptaken³⁵S and³²P was released at 5 °C. Longer exposure to a chilling temperature of 5 °C (1–7 days) resulted in two patterns of sulphate and phosphate uptake.     

Some factors affecting survival and oxygen uptake in a subtropical beach flea

The survival and oxygen uptake of the supralittoral amphipod Chroestia lota Marsden & Fenwick were investigated in humid air and sea water between 15 and 35°C. Seven-day exposure experiments were made on three size groups of amphipods at 6 constant temperatures (15, 20, 25, 30, 35, 40 °C) and three cyclic temperatures (15–25, 20–30, 25–35°C) in air and in sea water at 34 and 17%. salinity. Neither size, treatment nor temperature affected survival between 15 and 30°C. Mortality increased > 30°C with large individuals being consistently less tolerant than medium and small amphipods. While amphipods exposed to cyclic temperatures during submersion had reduced survival compared with constant temperatures, those individuals exposed to cyclic conditions in humid air showed the greatest resistance. Oxygen uptake of Chroestia increased with dry body wt and, over the range 15–35°C, this semi-terrestrial beach flea could maintain its aerial VO₂ following submersion. Oxygen uptake increased directly in proportion to gill area and the weight specific gill area was low, consistent with the need to reduce desiccation. It is suggested that total gill area does not limit oxygen uptake in Chroestia and that cutaneous respiration may be important especially in aquatic conditions.     

Leishmania donovani: effect of temperature on RNA metabolism

The present study concerns RNA metabolism of Leishmania donovani and its changes during exposure to higher temperatures. The incorporation of labeled precursors into RNA was found to be greatly decreased during incubation at 37 °C in comparison with that at the optimum temperature of growth (22 °C). The decreased incorporation was shown to be mainly due to augmented template RNA degradation. The RNA-polymerase activity remained unaffected at the elevated temperature, but the uptake of labeled uracil was markedly inhibited.     

Mechanism of colicin action: early events

The kinetics and the temperature dependence of potassium loss from Escherichia coli cells treated with colicin K have been examined. At 37 C, after a single lethal hit, essentially all of the intracellular potassium is lost within the first few minutes of treatment. The initial rate of loss is linearly related to colicin concentration up to a multiplicity of 30. As the temperature is decreased over the range from 37 to 1 C, an increasing delay is seen in the initiation of potassium loss after colicin adsorption. This delay can be overcome by increasing colicin multiplicity and probably reflects an alteration of the cell membrane at these temperatures. A comparison of this effect with an apparently related effect of temperature on the action of irehdiamine A indicates that the delay may represent the inhibition of a transmission process occurring in the membrane.     

Energy-dependent uptake of ochratoxin A by mitochondria.

The interaction of ochratoxin A, a mycotoxin produced by Aspergillus ochraceus, with isolated rat liver mitochondria and plasma membranes has been studied. Cell membranes bind [¹⁴C]ochratoxin A poorly and do not show saturation in the concentration range examined. The uptake of the toxin by mitochondria is saturable, with an apparent K_m at 0 °C of 30 nmol/mg of protein. Sonication or freeze-thawing reduces the extent of incorporation by 88%. Ochratoxin A uptake is energy dependent, resulting in a depletion of intramitochondrial ATP. Uncouplers such as m-chlorocarbonylcyanide phenylhydrazone or the respiratory inhibitors rotenone and antimycin A inhibit uptake 60–85%, while ATP reverses the antimycin and rotenone inhibition. Phosphate transport is sensitive to inhibition by the toxin, as measured by Ca²⁺ plus P_istimulated respiration and [³²P]P_i incorporation. In turn, phosphate inhibits nearly completely [¹⁴C]ochratoxin A uptake at 22 °C and causes a concomitant mitochondrial swelling yet is not incorporated into the matrix space. Thus, the saturable uptake of ochratoxin A is accompanied by a decrease in the energy state and inhibition of P_i transport, which results in deteriorative changes of the mitochondria, as evidenced by large-amplitude swelling.     

Pressure effects on the association of the and 2 subunits of tryptophan synthetase from Escherichia coli and Salmonella typhimurium

Sucrose density gradient centrifugation was employed to study the association of the α and β₂ subunits of the enzyme tryptophan synthetase from Escherichia coli and Salmonella typhimurium. In both cases, the fully associated enzyme (α₂β₂) showed a sedimentation coefficient of 6.4 S, in agreement with the values reported by other workers for the E. coli enzyme. The substrate, l-serine, and the cofactor, pyridoxal phosphate, were required for complex formation in both cases. Generation of moderately high pressures by increasing the centrifuge speed from 39,000 rpm to 50,000 rpm was found to interfere with complex formation in both species at 5 °C. This effect was reversed by a temperature increase from 5 °C to 20 °C or by low concentrations of a nonpolar solvent, ethanol, at 5 °C. These results indicate that hydrophobic bonding plays an important role in the formation of the active tryptophan synthetase α₂β₂ complex. Monovalent and divalent cations also interfered with the formation of the α₂β₂ complex, indicating the possibility that ionic bonds are also involved.     

The combined effects of temperature,salinity, and declining oxygen tension on oxygen consumption in the marine pulmonate Amphibola crenata (Gmelin, 1791)

Oxygen consumption of Amphibola crenata (Gmelin) was measured in various salinity-temperature combinations (< 0.1‰ to 41‰ salinity and 5 to 30°C) in air, and following exposure to declining oxygen tensions. In all experimental conditions, respiration varied with the 0.44 power of the body weight (sd = 0.14). The aquatic rate was consistently higher than the aerial rate of oxygen consumption, although at 30 °C the two rates were similar. Oxygen consumption increased with temperature up to 25 °C in all salinities; the lowest values were recorded at temperatures below 10 °C and at 30 °C in the most dilute medium. At all exposure temperatures, the oxygen consumption of Amphibola decreased regularly with salinity down to 0.1 ‰, and following exposure to concentrated sea water (41‰). Salinity had the least effect at 15 °C which was the acclimation temperature. In general, all of the temperature coefficients (Q₁₀ values) were low, < 1.65. However, Q₁₀ values above 2.8 were recorded at a salinity of 17.8‰ between 10 and 15 °C. Oxygen consumption of all size classes of Amphibola was more temperature dependent in air than in water and small individuals show a greater difference between their aerial and aquatic rates than larger snails. The rates of oxygen consumption in declining oxygen tensions were expressed as fractions of the rates in air saturated sea water at each experimental salinity-temperature combination. The quadratic coefficient B₂ becomes increasingly more negative with both decreasing salinity and temperatures up to 20 °C. At higher temperatures (25 and 30 °C) the response is reversed such that O₂ uptake in snails becomes increasingly independent of declining oxygen tensions at higher salinities. On exposure to a salinity of 4‰, Amphibola showed no systematic response to declining oxygen tension with respect to temperature. The ability of Amphibola to maintain its rate of oxygen consumption in a wide range of environmental conditions is discussed in relation to its potential for invading terrestrial habitats and its widespread distribution on New Zealand's intertidal mudflats.     

Effects of sodium ions on the electrical and pH gradients across the membrane of streptococcus lactis cells

Energized cells of Streptococcus lactis conserve and transduce energy at the plasma membrane in the form of an electrochemical gradient of hydrogen ions (Δp). An increase in energy-consuming processes, such as cation transport, would be expected to result in a change in the steady state Δp. We determined the electrical gradient (ΔΨ) from the fluorescence of a membrane potential-sensitive cyanine dye, and the chemical H⁺ gradient (ΔpH) from the distribution of a weak acid. In glycolyzing cells incubated at pH 5 the addition of NaCl to 200 mM partially dissipated the Δp by decreasing ΔΨ, while the ΔpH was constant. The Δp was also determined independently from the accumulation levels of thiomethyl-β-galactoside. The Δp values decreased in cell fermenting glucose at pH 5 or pH 7 when NaCl was added, while the ΔpH values were unaffected; cells fermenting arginine at pH 7 showed similar effects. Thus, these nongrowing cells cannot fully compensate for the energy demand of cation transport.     

Comparison of thermotolerance of sun-exposed peel and shaded peel of ‘Fuji’ apple

The thermotolerance of the sun-exposed peel and the shaded peel of ‘Fuji’ apple (Malus domestica Borkh.) fruit was evaluated by measuring pigments, chlorophyll a fluorescence transients and O₂ evolution or uptake after exposure to 25, 35, 40, 42, 44, 46 or 48 °C for 30 min in the dark. A major effect of heat stress at 46–48 °C on the chlorophyll a fluorescence transients was the appearance of a very clear K step at 200–300 μs for both peel types. The K step was slightly more pronounced in the sun-exposed peel than in the shaded peel, suggesting that the resistance of oxygen-evolving complex to heat stress is slightly lower in the sun-exposed peel than in the shaded peel. Minimal fluorescence (F_O), relative to the value at 25 °C, increased to a greater extent in the shaded peel than in the sun-exposed peel after exposure to 46–48 °C, but the temperature dependencies of F_O changes were similar for both peel types. Maximum quantum yield of PSII (F_V/F_M) decreased to a similar extent in the sun-exposed peel and the shaded peel as temperature rose from 25 to 44 °C, but the sun-exposed peel reached slightly lower values at 46–48 °C. Correspondingly, gross O₂ evolution rate, relative to that at 25 °C, was also slightly lower in the sun-exposed peel than in the shaded peel at 46–48 °C. In response to heat stress, the ratio of Q_A-reducing reaction centers (RCs) to total RCs and the ratio of Q_B-reducing RCs to Q_A-reducing RCs decreased, but both of them decreased to lower values in the sun-exposed peel than in the shaded peel at 46–48 °C, indicating that the capacity of electron transfer between P₆₈₀⁺ and Q_B via Q_A was damaged to a greater extent in the sun-exposed peel than in the shaded peel. At each given temperature, dark respiration was similar between the two peel types. Overall, it appears that the exposure to higher surface temperature under high light does not make the sun-exposed peel more tolerant of heat stress than the shaded peel of apple fruit.     

Adsorption of β-Lactoglobulin onto the Surface of Stainless Steel Particles

The adsorption of β-Lactoglobulin (β-Lg), one of the main constituents of fouling deposits in milk processing, onto the surface of stainless steel particles was studied under various conditions. The adsorption isotherm of β-Lg at 25°C was of the Langmuir type, and the plateau suggested that the surface was covered by a monolayer of β-Lg. The amount of β-Lg adsorbed steeply increased above 65°C. At 75°C, it increased almost linearly with the protein concentration in the bulk solution. Heating and chemical modification of the SH-group caused a much smaller amount of β-Lg to be adsorbed at 75°C. These findings indicate that the thermal aggregation of denatured β-Lg at the surface is important in the adsorption. More β-Lg was adsorbed at pH 4 than at pH 6.85. This suggests that the electrostatic interaction between β-Lg and the surface contributes to the adsorption behavior.     

Hydrophobic interactions of the apo and holo forms of human cobalamin binding proteins

The interactions of transcobalamin II (TC II), intrinsic factor (IF) and R-type binding protein of cobalamin (Cb1, vitamin B₁₂) with the hydrophobic chromatography matrix Phenyl-Sepharose CL-4B were investigated. IF-Cb1 and R-Cb1 complexes were not adsorbed on Phenyl-Sepharose at room temperature or at 4°C with buffer containing 50 mM sodium phosphate, pH 7.4 containing 150 mM sodium chloride. The TC II-Cb1 complex adsorbed and could be eluted with buffer containing 50% $\text{v}\text{v}$ glycerol. IF without Cb1 adsorbed and was eluted with 50% glycerol at room temperature and 4°C. At room temperature, R binder without Cb1 eluted with buffer, but later than the R-Cb1 complex. At 4°C, R binder completely adsorbed to the matrix. TC II-without Cb1 bound to the matrix at 4°C and room temperature and could not be eluted with glycerol. These results suggest that Cb1 binding proteins can be separated and identified based on their hydrophobic properties. In addition, upon binding Cb1, TC II, IF and R-type binders undergo a conformational change such that the protein-Cb1 complex shows reduced hydrophobicity.     

Effect of altered temperature storage on the in vitro cellular uptake of liposome drug products

The aim of this study was to evaluate whether temperature stress conditions affect the cellular uptake of liposomal doxorubicin, Doxil^® (DXL; Ortho Biotech, Raritan, New Jersey, USA), and liposomal daunorubicin, DaunoXome^® (DXM; Gilead Sciences, San Dimas, California, USA). Uptake of these cytotoxic compounds is essential for their pharmacological effect. Commercially available DXL and DXM were stressed for 6 days under altered temperature conditions of 22 and 50°C, as compared to storage in their buffered formulations at the labeled temperature of 4°C. The cellular uptake of the liposomal drugs was measured by fluorescence intensity in human ovarian SKOV-3 and murine macrophage J774A.1 cell lines following a 4-hour exposure to DXL or DXM. There was a 5- to 10-fold increase in the cellular uptake of DXL and DXM in both cell lines after stress exposure to 50°C. Exposure of DXL to 22°C stress decreased its uptake by SKOV-3 cells, when compared to exposure of DXL to 4°C control conditions. A cell-based uptake assay may provide a means to assess changes in the functional activity of liposomes in conjunction with evaluation of their physicochemical properties in order to evaluate the stability and integrity of liposomes.