Influence of membrane physical state on lysosomal potassium ion permeability

Lysosomal permeability to potassium ions is an important property of the organelle. Influence of the membrane physical state on the potassium ion permeability of isolated lysosomes was assessed by measuring the membrane potential with bis(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol and monitoring the lysosomal proton leakage with p-nitrophenol. The membrane fluidity of lysosomes was modulated by treatment with membrane fluidizer benzyl alcohol and rigidifier cholesteryl hemisuccinate. Changes in the membrane order were examined by steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene. The measurements of membrane potential and proton leakage demonstrated that the permeability of lysosomes to potassium ions increased with rigidification of their membranes by cholesteryl hemisuccinate treatment at 37 degrees C, and decreased with fluidization of their membranes by benzyl alcohol treatment at 2 degrees C. The changes in ion permeability could be recovered by fluidizing the rigidified membranes and rigidifying the fluidized membranes. The results suggest that the physical states of lysosomal membranes play an important role in the regulation of their K(+) permeability.     

Selective erythrocyte potassium efflux following pulse treatment with tellurite.

Human erythrocytes exposed to 0.1 mM tellurite (K2TeO3) in an isotonic buffered choline chloride medium for 15 min at 37 degrees C, washed, and incubated further in the absence of the chemical in the buffer, exhibited selective leakiness for potassium within minutes. The potassium efflux curve was sigmoidal, with an initially slow leakage followed by a sharp rise (first-order kinetics) and a plateau by 60 min. After 15 min, 30-50% of the total potassium concentration had leaked from the cells, although less than 1% lysis had occurred. The control cells incubated in buffer with no K2TeO3 exhibited no potassium leakage. The mean volume of the K2TeO3-treated erythrocytes increased and their median density decreased, indicating changes in the colloid osmotic state and physical characteristics of the cells. However, cells pretreated with K2TeO3 exhibited no significant change in glutathione (GSH) concentration and no membrane lipid peroxidation, unlike cells pretreated with t-butylhydroperoxide (Deuticke et al., Biochim. Bio phys. Acta, 899, 125-128, 1987). The enhanced potassium permeability of the K2TeO3-treated erythrocytes preceded the increase in cell volume, intracellular hydration, and a decrease in median density. We suggest that perturbation of the lipid-protein interaction in the membrane by the oxidant alters the potassium permeability and results in the selective leakage with eventual hemolysis.     

Cholesterol depletion induces solid-like regions in the plasma membrane

Glycosylphosphatidylinositol-linked and transmembrane major histocompatibility complex (MHC) class II I-E(k) proteins, as well as N-(6-tetramethylrhodaminethiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (Tritc-DHPE), are used as probes to determine the effect of cholesterol concentration on the organization of the plasma membrane at temperatures in the range 22 degrees C-42 degrees C. Cholesterol depletion caused a decrease in the diffusion coefficients for the MHC II proteins and also for a slow fraction of the Tritc-DHPE population. At 37 degrees C, reduction of the total cell cholesterol concentration results in a smaller suppression of the translational diffusion for I-E(k) proteins (twofold) than was observed in earlier work at 22 degrees C (five sevenfold) Vrljic, M., S. Y. Nishimura, W. E. Moerner, and H. M. McConnell. 2005. Biophys. J. 88:334-347. At 37 degrees C, the diffusion of both I-E(k) proteins is Brownian (0.9 < alpha-parameter < 1.1). More than 99% of the protein population diffuses homogeneously when imaged at 65 frames per s. As the temperature is raised from 22 degrees C to 42 degrees C, a change in activation energy is seen at approximately 35 degrees C in the Arrhenius plots. Cytoskeletal effects appear to be minimal. These results are consistent with a previously described model of solid-like domain formation in the plasma membrane.     

Membrane radiosensitivity of fatty acid supplemented fibroblasts as assayed by the loss of intracellular potassium

Leakage of potassium from mouse fibroblast LM cells, X-irradiated at 0 degrees C with doses up to 400 Gy is shown to be related to plasma membrane lipid composition. Fatty acid supplemented cells, containing about 40 per cent polyunsaturated fatty acids (PUFA) in their membranes were much more sensitive to radiation, as measured by increased permeability, than normal cells, which contained 7 per cent PUFA. The damage observed after irradiation at 0 degrees C was partially repaired during a post-irradiation incubation at 22 degrees C. The o.e.r. for potassium leakage was about 4 for normal fibroblasts and 8 for the PUFA-supplemented cells. No oxygen-dependent radiation damage could be observed in cells treated with high amounts of vitamin E. Depletion of glutathione in PUFA cells sensitized oxic cells to radiation damage, resulting in an increase of the o.e.r. from 8 to 17. No lipid peroxidation (malondialdehyde production and disappearance of fatty acyl chains) could be demonstrated. While PUFA, normal and vitamin E grown cells showed a differential sensitivity in radiation-induced potassium leakage and trypan blue uptake (high doses, interphase death), no difference in radiation-induced clonogenic ability (reproductive death) could be observed after the different cell treatments. The experiments reported are supportive of a role of membranes in the mechanism of radiation-induced interphase death and show that increased damage may be expected when high amounts of polyunsaturated membrane lipids are present under conditions of low amounts of appropriate antioxidants.     

Yeast secretory mutants that block the formation of active cell surface enzymes

Yeast cells secrete a variety of glycosylated proteins. At least two of these proteins, invertase and acid phosphatase, fail to be secreted in a new class of mutants that are temperature-sensitive for growth. Unlike the yeast secretory mutants previously described (class A sec mutants; Novick, P., C. Field, and R. Schekman, 1980, Cell., 21:205-420), class B sec mutants (sec 53, sec 59) fail to produce active secretory enzymes at the restrictive temperature (37 degrees C). sec 53 and sec 59 appear to be defective in reactions associated with the endoplasmic reticulum. Although protein synthesis continues at a nearly normal rate for 2 h at 37 degrees C, incorporation of [3H]mannose into glycoprotein is reduced. Immunoreactive polypeptide forms of invertase accumulate within the cell which have mobilities on SDS PAGE consistent with incomplete glycosylation: sec 53 produces little or no glycosylated invertase, and sec 59 accumulates forms containing 0-3 of the 9-10 N-linked oligosaccharide chains that are normally added to the protein. In addition to secreted enzymes, maturation of the vacuolar glycoprotein carboxypeptidase Y, incorporation of the plasma membrane sulfate permease activity, and secretion of the major cell wall proteins are blocked at 37 degrees C.     

Micro-scission of YAC tumor cells during antibody-dependent cellular cytotoxicity mediated by human neutrophils

The cellular events accompanying neutrophil-mediated antibody-dependent cellular cytotoxicity (ADCC) directed against YAC erythroleukemic target cells have been studied by time-lapse fluorescence-intensified microscopy. The YAC plasma membrane and cytosol were labeled with the fluorescent probes diC18Icc and eosin Y, respectively. Fluorescently labeled and IgG-opsonized YAC cells were incubated at 37 degrees C while observed by optical microscopy. During temporal studies of neutrophil-YAC conjugates, the cytosol of YAC cells accumulated in tubular and spherical compartments of the neutrophils' vacuolar apparatuses. To distinguish between several possible mechanisms of target cytosol uptake, diC18Icc-labeled YAC cells were observed during identical conditions. The membrane label diC18Icc was found to accumulate within neutrophils in an identical fashion. At roughly 30 min, 25 and 38% of neutrophils in apparent conjugates had internalized tumor cell cytosol or plasma membrane, respectively, within a vesicular compartment. The IgG-dependent uptake of eosin Y and diC18Icc by neutrophils was diminished by exposure to 2.5 mM sodium azide. When cells were exposed to 5.5 mM sodium azide, 1 mM iodoacetamide, or 4 degrees C, conjugate formation and uptake of eosin Y or diC18Icc were abolished. An artifactual accumulation of eosin Y or diC18Icc in neutrophils was further ruled out by control studies. Non-specific exchanges of eosin Y and diC18Icc labels of YAC cells with tannic acid-treated red blood cells (RBCs) and normal neutrophils were studied. Since hemoglobin binds tightly to eosin Y, RBCs can easily detect eosin Y leakage. No exchange of eosin Y or diC18Icc from YAC cells into bound tannic acid-treated erythrocytes was found.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationship of hyperthermia-induced hemolysis of human erythrocytes to the thermal denaturation of membrane proteins

Hemolysis of human erythrocytes as a function of time of exposure to 47.4-54.5 degrees C was measured and correlated to thermal transitions in the membranes of intact erythrocytes as determined by differential scanning calorimetry (DSC). Curves of hemoglobin leakage (a measure of hemolysis) as a function of time have a shoulder region exhibiting no leakage, indicative of the ability to accumulate sublethal damage (i.e., damage not sufficient to cause lysis), followed by a region of leakage approximating pseudo-first-order kinetics. Inverse leakage rates (Do) of 330-21 min were obtained from 47.4-54.5 degrees C, respectively. A relatively high activation energy of 304 +/- 22 kJ/mol was obtained for leakage, eliminating the involvement of metabolic processes but implicating a transition as the rate-limiting step. Membrane protein involvement was suggested by the very low rate (10(-2) of the rate from erythrocytes) and low activation energy (50 +/- 49 kJ/mol) of hemoglobin leakage from liposomes containing no membrane protein. A model was developed that predicts a transition temperature (Tm) for the critical target (rate-limiting step) of 60 degrees C when measured at a scan rate of 1 K/min. DSC scans were obtained from intact erythrocytes and a procedure developed to fit and remove the transition for hemoglobin denaturation which dominated the scan. Three transitions remained (transitions A, B, and C) with Tm values of 50.0, 56.8, and 63.8 degrees C, respectively. These correspond to, but occur at slightly different temperatures than, the A, B, and C transitions of isolated erythrocyte membranes in the same salt solution (Tm = 49.5, 53-58, and 65.5 degrees C, respectively). In addition, the relative enthalpies of the three transitions differ between isolated membranes and erythrocytes, suggestive of membrane alterations occurring during isolation. Thus, all analyses were conducted on DSC scans of intact erythrocytes. The B transition is very broad and probably consists of several transitions. An inflection, which is seen as a distinct peak (transition B3) in fourth-derivative curves, occurs at 60.8 degrees C and correlates well with the predicted Tm of the critical target. Ethanol (2.2%) lowers the Tm of B3 by 4.0-4.5 K, close to the shift of 3.3 K predicted from its effect on hemolysis. Glycerol (10%) has very little effect on both hemolysis and the Tm of B3, but it stabilizes spectrin (delta Tm = 1.5 K) against thermal denaturation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Defective assembly of a hybrid vacuolar H(+)-ATPase containing the mouse testis-specific E1 isoform and yeast subunits

Mammalian vacuolar-type proton pumping ATPases (V-ATPases) are diverse multi-subunit proton pumps. They are formed from membrane V(o) and catalytic V(1) sectors, whose subunits have cell-specific or ubiquitous isoforms. Biochemical study of a unique V-ATPase is difficult because ones with different isoforms are present in the same cell. However, the properties of mouse isoforms can be studied using hybrid V-ATPases formed from the isoforms and other yeast subunits. As shown previously, mouse subunit E isoform E1 (testis-specific) or E2 (ubiquitous) can form active V-ATPases with other subunits of yeast, but E1/yeast hybrid V-ATPase is defective in proton transport at 37 degrees C (Sun-Wada, G.-H., Imai-Senga, Y., Yamamoto, A., Murata, Y., Hirata, T., Wada, Y., and Futai, M., 2002, J. Biol. Chem. 277, 18098-18105). In this study, we have analyzed the properties of E1/yeast hybrid V-ATPase to understand the role of the E subunit. The proton transport by the defective hybrid ATPase was reversibly recovered when incubation temperature of vacuoles or cells was shifted to 30 degrees C. Corresponding to the reversible defect of the hybrid V-ATPase, the V(o) subunit a epitope was exposed to the corresponding antibody at 37 degrees C, but became inaccessible at 30 degrees C. However, the V(1) sector was still associated with V(o) at 37 degrees C, as shown immunochemically. The control yeast V-ATPase was active at 37 degrees C, and its epitope was not accessible to the antibody. Glucose depletion, known to dissociate V(1) from V(o) in yeast, had only a slight effect on the hybrid at acidic pH. The domain between Lys26 and Val83 of E1, which contains eight residues not conserved between E1 and E2, was responsible for the unique properties of the hybrid. These results suggest that subunit E, especially its amino-terminal domain, plays a pertinent role in the assembly of V-ATPase subunits in vacuolar membranes.     

The population variability of Yersinia pestis in soil samples from the natural focus of plague

Three Y. pestis strains were found to exist in the experimental soil ecosystem at a temperature of 4 degrees - 8 degrees C for a longer period (10 months, the term of observation) than at room temperature (3.5 months). Y. pestis population structure was characterized by relative stability in strains of the subspecies altaica and heterogeneity in the strain of the main subspecies, manifested by the loss of the pgm locus by vegetative cells and the preservation of pgm+ variants in the latent (uncultivable) form (LF). In the populations of all strains uniformity in calcium dependence, the tendency towards a decrease in the synthesis of factor 1, nutritional requirements in amino acids was observed. An important factor of the preservation of Y. pestis in the soil was LF formation. At room temperature this process quickly resulted in the death of the population. At 4 degrees - 8 degrees C A. pestis altaica avirulent strain could be inoculated onto solid nutrient media for a two-fold longer period (for 4 month) than the strain with selective virulence and for 5.5 months longer than Y. pestis pestis highly virulent strain.     

Ninth component of complement: self-aggregation and interaction with lipids

We have investigated environmental conditions that might be of importance for the polymerization of the ninth component (C9) of human complement. In disagreement with earlier reports summarized by Tschopp et al. [Tschopp, J., Müller-Eberhard, H. J., & Podack, E. R. (1982) Nature (London) 298, 534-538] we find no evidence for significant aggregation or loss of hemolytic activity of C9 when incubated at 37 degrees C even after 12 days of incubation. Higher temperatures cause denaturation of the protein and formation of stringlike aggregates. In contrast, short-term proteolysis with 1% (w/w) trypsin at room temperature causes rapid polymerization of part of the C9 into tubular structures (poly-C9), and the remainder of the monomeric C9 is digested. This polymerization reaction is inhibitable by trypsin inhibitor; alpha-thrombin and proteinase K are ineffective in creating polymers. A second discrepancy to the earlier reports is our finding that monomeric C9 immediately interacts with small unilamellar lipid vesicles (SUV) without a required heating step. As a result of this interaction about half of the C9 aggregates to form strings and tubules, and these aggregates cause agglutination of vesicles. The other half of the C9 associates with a second population of SUV without causing a change in Stokes' radius of these vesicles, and no proteinaceous structures are detectable on the vesicle surface by electron microscopy. When these two vesicle populations are tested for their membrane integrity, no release of an encapsulated fluorescent marker can be detected, nor is there leakage of potassium ions across the bilayer membrane since a membrane diffusion potential can be developed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium-dependent and phosphorylation-stimulated proteolysis of lipocortin I by an endogenous A431 cell membrane protease

Purified placental lipocortin I but not lipocortin II was proteolyzed during A431 cell membrane-catalyzed phosphorylation reactions. Proteolysis was Ca2+-dependent but was not prevented in the presence of a variety of inhibitors of Ca2+-dependent proteases, suggesting that the Ca2+ effect is a property of lipocortin I itself. Proteolysis was inhibited by Triton X-100 or dithiothreitol and was temperature-dependent, occurring at 30 degrees C but not at 0 degrees C. Tyrosine phosphorylation and proteolysis are distinct events as both phosphorylated and nonphosphorylated lipocortins could be cleaved by the membrane protease, but prephosphorylation enhanced the rate of proteolysis 2-fold during the initial reaction and by 60 min almost half of the phosphorylated lipocortin was proteolyzed. Cleavage of the 38-kDa phosphotyrosyl lipocortin I generated a truncated 37-kDa form of lipocortin which retained the phosphate label, indicating that proteolysis occurred at a site N-terminal to the site of tyrosine phosphorylation, possibly at tryptophan 12. Ando, Y., Imamura, S., Hong, Y.-M., Owada, M.K., Kakunaga, T., and Kannagi, R. [1989) J. Biol. Chem. 264, 6948-6955) have recently reported that in vitro cleavage at sites in the N-terminal tail region of lipocortin I by exogenously added proteases dramatically enhanced the Ca2+ sensitivity of phospholipid binding by lipocortin. The demonstrated ability of an endogenous membrane protease to catalyze a similar and specific cleavage in a Ca2+-dependent manner indicates that this event may occur in the cell where it would have important effects on the functional properties of lipocortin I.     

Thermal adaptation of Tetrahymena membranes with special reference to mitochondria. II. Preferential interaction of cardiolipin with specific molecular species of phospholipid

A specific effect of cardiolipin on fluidity of mitochondrial membranes was demonstrated in Tetrahymena cells acclimated to a lower temperature in the previous report (Yamauchi, T., Ohki, K., Maruyama, H. and Nozawa, Y. (1981) Biochim. Biophys. Acta 649, 385-392). This study was further confirmed by the experiment using fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH). Anisotropy of DPH for microsomal and pellicular total lipids from Tetrahymena cells showed that membrane fluidity of these lipids increased gradually as the cells were incubated at 15 degrees C after the shift down of growth temperature from 39 degrees C. However, membrane fluidity of mitochondrial total lipids was kept constant up to 10 h. This finding is compatible with the result obtained using spin probe in the previous report. Additionally, the break-point temperature of DPH anisotropy was not changed in mitochondrial lipids whereas those temperatures in pellicular and microsomal lipids lowered during the incubation at 15 degrees C. Interaction between cardiolipins and various phospholipids, which were isolated from Tetrahymena cells grown at 39 degrees C or 15 degrees C and synthesized chemically, was investigated extensively using a spin labeling technique. The addition of cardiolipins from Tetrahymena cells grown at either 39 degrees C or 15 degrees C did not change the membrane fluidity (measured at 15 degrees C) of phosphatidylcholine from whole cells grown at 39 degrees C. On the other hand, both cardiolipins of 39 degrees C-grown and 15 degrees C-grown cells decreased the membrane fluidity of phosphatidylcholine from Tetrahymena cells grown at 15 degrees C. The same results were obtained for phosphatidylcholines of mitochondria and microsomes. Membrane fluidity of phosphatidylethanolamine, isolated from cells grown at 15 degrees C, was reduced to a small extent by Tetrahymena cardiolipin whereas that of 39 degrees C-grown cells was not changed. Representative molecular species of phosphatidylcholines of cells grown at 39 degrees C and 15 degrees C were synthesized chemically; 1-palmitoyl-2-oleoylphosphatidylcholine for 39 degrees C-grown cells and dipalmitoleoylphosphatidylcholine for 15 degrees C-grown ones. By the addition of Tetrahymena cardiolipin, the membrane fluidity of 1-palmitoyl-2-oleoylphosphatidylcholine was not changed but that of dipalmitoleoylphosphatidylcholine was decreased markedly. These phenomena were caused by Tetrahymena cardiolipin. However, bovine heart cardiolipin, which has a different composition of fatty acyl chains from the Tetrahymena one, exerted only a small effect.     

Calcium currents in a fast-twitch skeletal muscle of the rat

Slow ionic currents were measured in the rat omohyoid muscle with the three-microelectrode voltage-clamp technique. Sodium and delayed rectifier potassium currents were blocked pharmacologically. Under these conditions, depolarizing test pulses elicited an early outward current, followed by a transient slow inward current, followed in turn by a late outward current. The early outward current appeared to be a residual delayed rectifier current. The slow inward current was identified as a calcium current on the basis that (a) its magnitude depended on extracellular calcium concentration, (b) it was blocked by the addition of the divalent cations cadmium or nickel, and reduced in magnitude by the addition of manganese or cobalt, and (c) barium was able to replace calcium as an inward current carrier. The threshold potential for inward calcium current was around -20 mV in 10mM extracellular calcium and about -35 mV in 2 mM calcium. Currents were net inward over part of their time course for potentials up to at least +30 mV. At temperatures of 20-26 degrees C, the peak inward current (at approximately 0 mV) was 139 +/- 14 microA/cm2 (mean +/- SD), increasing to 226 +/- 28 microA/cm2 at temperatures of 27-37 degrees C. The late outward current exhibited considerable fiber-to-fiber variability. In some fibers it was primarily a time-independent, nonlinear leakage current. In other fibers it was primarily a time-independent, nonlinear leakage current. In other fibers it appeared to be the sum of both leak and a slowly activated outward current. The rate of activation of inward calcium current was strongly temperature dependent. For example, in a representative fiber, the time-to-peak inward current for a +10-mV test pulse decreased from approximately 250 ms at 20 degrees C to 100 ms at 30 degrees C. At 37 degrees C, the time-to-peak current was typically approximately 25 ms. The earliest phase of activation was difficult to quantify because the ionic current was partially obscured by nonlinear charge movement. Nonetheless, at physiological temperatures, the rate of calcium channel activation in rat skeletal muscle is about five times faster than activation of calcium channels in frog muscle. This pathway may be an important source of calcium entry in mammalian muscle.     

Fusion of phosphatidylethanolamine-containing liposomes and mechanism of the L alpha-HII phase transition

The initial kinetics of fusion and leakage of liposomes composed of N-methylated dioleoylphosphatidylethanolamine (DOPE-Me) have been correlated with the phase behavior of this lipid. Gagné et al. [Gagné, J., Stamatatos, L., Diacovo, T., Hui, S. W., Yeagle, P., & Silvius, J. (1985) Biochemistry 24, 4400-4408] have shown that this lipid is lamellar (L alpha) below 20 degrees C, is hexagonal (HII) above 70 degrees C, and shows isotropic 31P NMR resonances at intermediate temperatures. This isotropic state is also characterized by complex morphological structures. We have prepared DOPE-Me liposomes at pH 9.5 and monitored the temperature dependence of the mixing of aqueous contents, leakage, and changes in light scattering upon reduction of the pH to 4.5. At and below 20 degrees C, where the lipid is in the L alpha phase, there is very little aggregation or destabilization of the liposomes. Between 30 and 60 degrees C, i.e., where the lipid is in the isotropic state, the initial rates of liposome fusion (mixing of aqueous contents) and leakage increase. At temperatures approaching that where the hexagonal HII phase transition occurs, the initial rates and extents of fusion decrease, whereas leakage is enhanced. Similar results were found for dioleoylphosphatidylethanolamine/dioleoylphosphatidylcholine (2:1) liposomes. These results clearly establish a common mechanism between the appearance of the isotropic state (between the L alpha and HII phases) and the promotion of liposome fusion. We propose a simple model to explain both the observed behavior of phosphatidylethanolamine-containing membranes with respect to liposome fusion and/or lysis and the beginning of the L alpha-HII phase transition.     

Rat brain membrane-bound delta opioid receptor: loss and reactivation of binding on dialysis and aging at low temperature.

A change in the environment of rat brain membranes by dialysis from phosphate buffered saline (PBS) to 10 mM potassium phosphate (pH 7.2) led to a 35% loss in delta opioid receptor binding, while alteration of membrane structure on freezing at -20 degrees C for 55 days led to 85% loss of receptor binding. The dialysate, 200 mM KCI and NaCl restored receptor binding lost on dialysis. This K+ and Na+ restabilization of the receptor can be through cation-pi bonding, interactions that are suited to the lipid bilayer. In membranes stored at -20 degrees C, the loss of binding is attributed to increased membrane fluidity by phospholipase A2 action on membrane phospholipids, resulting in an increase of free fatty acids. K+ but not Na+ restabilization of these membrane receptors may be due to the ability of K+ to decrease membrane fluidity.     

cDNA cloning and characterization of temperature-acclimation-associated light meromyosins from grass carp fast skeletal muscle

The three types of cDNA clones, previously defined as the 10 degrees C, intermediate and 30 degrees C-types [Tao, Y., Kobayashi, M., Liang, C.S., Okamoto, T., Watabe, S., 2004. Temperature-dependent expression patterns of grass carp fast skeletal myosin heavy chain genes. Comp. Biochem. Physiol. B 139, 649-656], were determined for their 5'-regions which encoded at least the C-terminal half of myosin rod, light meromyosin (LMM), in fast skeletal muscles of grass carp Ctenopharyngodon idella. The deduced amino acid sequence identity was 91.1% between the 10 degrees C and 30 degrees C-types and 91.4% between the 10 degrees C and intermediate-types, whereas a high sequence identity of 97.8% was found between the intermediate and 30 degrees C-types. These three grass carp LMMs all had a characteristic seven-residue (heptad) repeat (a, b, c, d, e, f, g)(n), where positions a and d were normally occupied by hydrophobic residues, and positions b, c and f by charged residues. However, the ratios of hydrophobic residues to the total were higher for the intermediate- and 30 degrees C- than 10 degrees C-type LMM, suggesting that the former both types may form more stable coiled-coils of alpha-helices than the latter type. These differences in the primary structures of LMM isoforms might be partially implicated in differences in the thermostabilities and gel-forming profiles of myosins from grass carp in different seasons reported previously [Tao, Y., Kobayashi, M., Fukushima, H., Watabe, S., 2005. Changes in enzymatic and structural properties of grass carp fast skeletal myosin induced by the laboratory-conditioned thermal acclimation and seasonal acclimatization. Fish. Sci. 71, 195-204; Tao, Y., Kobayashi, M., Fukushima, H., Watabe, S., 2007. Changes in rheological properties of grass carp fast skeletal myosin induced by seasonal acclimatization. Fish. Sci. 73, 189-196].     

Cytotoxic and genotoxic effects of tert-butyl hydroperoxide on Chinese hamster B14 cells

The organic hydroperoxide, tert-butyl hydroperoxide (t-BHP), is a useful model compound to study mechanisms of oxidative cell injury. In the present work, we examined the features of the interactions of this oxidant with Chinese hamster B14 cells. The aim of our study was to reveal a possible role of structural modifications in membranes and loss of DNA integrity in t-BHP-induced cell injury and death. The tert-butyl hydroperoxide treatment (100-1000 microM, 37 degrees C for 1h) did not decrease cell viability (as measured by cell-specific functional activity with an MTT test), but completely prevented cell growth. We observed intracellular reduced glutathione (GSH) oxidation and total glutathione (GSH+GSSG) depletion, a slight increase in the level of lipid-peroxidation products, an enhancement of membrane fluidity, intracellular potassium leakage and a significant decrease of membrane potential. At oxidant concentrations of 100-1500 microM, a significant damage to DNA integrity was observed as revealed by the Comet assay. The inhibition of cell proliferation (cell-growth arrest) may be explained by genotoxicity of t-BHP, by disturbance of the cellular redox-equilibrium (GSH oxidation) and by structural membrane modifications, which result in ion-non-selective pore formation. The disturbance in passive membrane permeability and the DNA damage may be the most dramatic cell impairments induced by t-BHP treatment. The presence of another oxidant, hypochlorous acid (HOCl), completely prevented t-BHP-induced DNA strand breaks, perhaps due to extracellular oxidation of t-BHP by HOCl.     

Transient-phase studies on the creatine kinase reaction. The analysis of a reaction pathway with three intermediates.

1. The initial formation of creatine phosphate by creatine kinase was studied in the millisecond range and the effect of temperature on the transient and steady-state phases exploited. 2. At 25 degrees C and 35 degrees C there was no transient phase. This is in agreement with the results of Gutfreund [Engelborghs, Y., Marsh, A., and Gutfreund, H. (1975) Biochem. J. 151, 47--50]. 3. At 4 degrees C the time course of creatine phosphate formation was complex and consisted of three transient phases: a lag phase, a burst phase and a steady-state phase. Based on this result a reaction scheme for creatine kinase which includes three intermediates was proposed. Despite the completeness of the time course, the extraction of estimates for the rate constants was difficult and computer simulation and iterative methods had to be resorted to. 4. Attempts were made to provide evidence for the complex enzyme.ADP.metaphosphate.creatine on the creatine kinase reaction pathway [cf. Milner-White, E.J. and Watts, D.C. (1971) Biochem. J. 122, 727--740]. Under the conditions used these attempts were unsuccessful at times down to 2.5 ms, at 4 degrees C or 35 degrees C.     

Interactive effects of soil temperature and moisture on Concord grape root respiration

Root respiration has important implications for understanding plant growth as well as terrestrial carbon flux with a changing climate. Although soil temperature and soil moisture often interact, rarely have these interactions on root respiration been studied. This report is on the individual and combined effects of soil moisture and temperature on respiratory responses of single branch roots of 1-year-old Concord grape (Vitis labruscana Bailey) vines grown in a greenhouse. Under moist soil conditions, root respiration increased exponentially to short-term (1 h) increases in temperature between 10 degrees C and 33 degrees C. Negligible increases in root respiration occurred between 33 degrees C and 38 degrees C. By contrast to a slowly decreasing Q10 from short-term temperature increases, when roots were exposed to constant temperatures for 3 d, the respiratory Q10 between 10 degrees C and 30 degrees C diminished steeply with an increase in temperature. Above 30 degrees C, respiration declined with an increase in temperature. Membrane leakage was 89-98% higher and nitrogen concentration was about 18% lower for roots exposed to 35 degrees C for 3 d than for those exposed to 25 degrees C and 15 degrees C. There was a strong interaction of respiration with a combination of elevated temperature and soil drying. At low soil temperatures (10 degrees C), respiration was little influenced by soil drying, while at moderate to high temperatures (20 degrees C and 30 degrees C), respiration exhibited rapid declines with decreases in soil moisture. Roots exposed to drying soil also exhibited increased membrane leakage and reduced N. These findings of acclimation of root respiration are important to modelling respiration under different moisture and temperature regimes.