Skeletal muscle pH dynamics during arousal from hibernation measured by31P NMR spectroscopy

Summary The time-course of changes in skeletal muscle pH during arousal from hibernation in the Columbian ground squirrel was studied using³¹P NMR spectroscopy. In hibernation (T _re 7–9°C), shoulder/neck muscle pH was 7.45±0.03 and _Im was 0.60. In euthermia (T _re 37°C), muscle pH was 7.24±0.05 and _Im was 0.75. Thus the overall pH-temperature coefficient was-0.009 pH units/°C, indicating acidification of the muscle in hibernation. During the transition from hibernation to euthermia, however, the muscle shows a nonlinear pattern of pH change. In early arousal (T _sh<20–25°C,T _re<15°C) muscle pH does not change and muscle _Im increases to 0.72. In later arousal (T _sh>20–25°C,T _re>15°C) muscle pH decreases gradually toward the euthermic value and muscle _Im increases only slightly from 0.72 to 0.75. These results support the hypothesis that intracellular acidification of the muscle, present during hibernation, is reversed in early arousal. This may facilitate an increase in muscle metabolism and the contribution of maximal shivering thermogenesis to rewarming of the animal.Abbreviations _Im dissociation ratio of protein imidazole buffergroups - NST non-shivering thermogenesis - BAT brown adipose tissue - dp H/dT temperature coefficient of pH - pH _i intracellular pH - ³¹ P NMR ³¹Phosphorus nuclear magnetic resonance - P _i chemical shift of inorganic phosphate relative to PCr - PCr phosphocreatine - T _b body temperature - T _re rectal temperature - T _sh subcutaneous shoulder temperature - T _a ambient temperature     

Lipid peroxidation as the mechanism of modification of brain 5′-nucleotidase activity in vitro

The effect of lipid peroxidation on the Mg²⁺-independent and Mg²⁺-dependent activity of brain cell membrane 5-nucleotidase was determined and the affinity of the active sites of Mg²⁺-dependent enzyme for 5-AMP (substrate) and Mg²⁺ (activator) was examined. Brain cell membranes were peroxidized at 37°C in the presence of 100 M ascorbate and 25 M FeCl₂ (resultant) for 10 min. The activity of 5-nucleotidase and lipid peroxidation products (thiobarbituric acid reactive substances) were determined. At 10 min, the level of lipid peroxidation products increased from 0.20±0.10 to 17.5±1.5 nmoles malonaldehyde/mg membrane protein. The activity of Mg²⁺-independent 5-nucleotidase increased from 0.201±0.020 in controls to 0.305±0.028 mol Pi/mg protein/hr in peroxidized membranes. In the presence of 10mM Mg²⁺, the activity increased by 5.8-fold in the peroxidized membrane preparation in comparison to 14-fold in control In peroxidized preparation, the affinity of active site of Mg²⁺-dependent 5-nucleotidase for 5-AMP tripled, as indicated by a significant decrease inK _m (K _m=95±2 M AMP for control;K _m=32±2 MAMP for peroxidized).V _max was significantly reduced from 3.35±0.16 in control to 1.70±.09 moles Pi/mg protein in peroxidized membranes. The affinity of the active site for Mg²⁺ significantly increased (K _m=6.17±0.37 mM Mg²⁺ for control;K _m=4.0±0.31 peroxidized). The data demonstrate that lipid peroxidation modifies the Mg²⁺-dependent 5-nucleotidase function by altering the active sites for both the substrate and the activator. The modification of the 5-nucleotidase activity and the loss of Mg²⁺-dependent activation observed in this in-vitro study are similar to the changes previously observed by us in the hypoxic brain in-vivo. This suggests that lipid peroxidation which specifically alters the active site may be the underlying mechanism of the modification of 5-nucleotidase during hypoxia.     

Phase transitions in plant cuticles

The effect of temperature on wet plant cuticles has been investigated with the following techniques: Calorimetry, densitometry, spin-label electron-spin-resonance-(ESR)-spectroscopy, photo bleaching, and light and electron microscopy. At low temperatures cuticles ofCitrus aurantium L. andHedera helix show, at 16.3°C, a sharp transition (T0.5°C) with a latent heat of 4.7±0.5 J g^-1-cuticle. Below transition: The main orientation of the polymer matrix is parallel to the normal of the cuticle and the main orientation of the layer with soluble lipids is perpendicular to the normal. The cuticle is in a rigid state. Above transition (between 16.3°C and 38°C): Only the orientation of the polymer matrix has changed (tilted in parts). There exist several very sharp (T0.1°C) transitions (38°C, 41°C, 45°C, 49°C, ...) with a latent heat in the order of 0.4 J g^-1-cuticle. Above 38°C: The lamella of the soluble lipids is in a fluid state. Above 45°C there is a change in the molecular orientation of the soluble lipids as well as in the polymer matrix. The soluble lipids are mainly oriented parallel to the normal. The dry cuticles show no phase transition between 0°C and 200°C. At room temperature a dry/wet transition can be observed.Abbreviations ESR-spectroscopy electron-spin-resonance-spectroscopy     

CCCP activation of the reconstituted NaK-pump

Summary In the NaK-ATPase proteoliposomes (PLs), the NaK-pump activity, Na⁺ uptake, and ATP hydrolysis were apparently enhanced by carbonyl cyanidem-chlorophenylhydrazone (CCCP) and other ionophores without ion gradients. These ionophore effects were not cation specific. Without ionophores, the PL's ATPase activity fell to its steady-state value within 3 sec at 15°C. This decrease in activity disappeared in the presence of CCCP. Since CCCP is believed to enhance proton mobility across the lipid bilayer and dissipate membrane potential (V _m ), we postulated that aV _m build-up partially inhibits the PLs by changing the conformation of the NaK-pump, and that CCCP eliminated this partial inhibition. Since this activation required extracellular K⁺ and high ATP concentration in the PLs, CCCP must affect the conversion between the phosphorylated forms of NaK-ATPase (EP); this step has been suggested by Goldschlegger et al. (1987) to be the voltage-sensitive step (J. Physiol. (London) 387:331–355). Although cytoplasmic K⁺ accelerated the change of ADP-and K⁺-sensitive EP (E^*P) to K⁺-sensitive ADP-insensitive EP (E₂P), CCCP did not compete with cytoplasmic K⁺ when cytoplasmic Na⁺ was saturated. When the PLs were phosphorylated with 20 m ATP and 20 m palmitoyl CoA instead of with high concentration of ATP, CCCP increased the E^*P content and decreased the ADP-sensitive K⁺-insensitive EP (E₁P). The results described above suggest that CCCP affects the E₁P to E^*P change in the E₁PE^*PE₂P conversion and that this reaction step is inhibited byV _m .     

Electric field-induced breakdown of lipid bilayers and cell membranes: A thin viscoelastic film model

Summary A simple viscoelastic film model is presented, which predicts a breakdown electric potential having a dependence on the electric pulse length which approximates the available experimental data for the electric breakdown of lipid bilayers and cell membranes (summarized in the reviews of U. Zimmermann and J. Vienken, 1982,J. Membrane Biol. 67:165 and U. Zimmermann, 1982,Biochim. Biophys. Acta 694:227). The basic result is a formula for the time of membrane breakdown (up to the formation of pores): =(/C)/( _m ² ₀ ² U ⁴/24Gh ³+T ²/Gh–1), where is a proportionality coefficient approximately equal to ln(h/2₀),h being the membrane thickness and ₀ the amplitude of the initial membrane surface shape fluctuation ( is usually of the order of unity), represents the membrane shear viscosity,G the membranes shear elasticity modules, _m the membrane relative permittivity, ₀=8.85×10^–12 Fm,U the electric potential across the membrane, the membrane surface tension andT the membrane tension. This formula predicts a critical potentialU _c ;U _c =(24Gh ³/ _m ² ₀ ² )^1/4 (for = andT=0). It is proposed that the time course of the electric field-induced membrane breakdown can be divided into three stages: (i) growth of the membrane surface fluctuations, (ii) molecular rearrangements leading to membrane discontinuities, and (iii) expansion of the pores, resulting in the mechanical breakdown of the membrane.     

Phenotypic instability in a tif-1 mutant of Escherichia coli

Summary The mutant T44() of Escherichia coli K12, grown in the presence of adenine, develops an increased tolerance to streptomycin. In cultures grown on streptomycin, the ts character (tif) may temporarily be suppressed but, on further transfer, both the temperature-sensitive phenotype and streptomycin tolerance disappear. In a cell-free system, the relative efficiency of translation of MS2 and poly U messenger RNAs was, respectively, 75 and 50% lower in extracts from cultures grown at 37° with adenine than in extracts from 30° cultures. Similar results were obtained when adenine was added in vitro to an extract from a culture grown at 37° in the absence of adenine, using MS2 RNA as messenger. Moreover, the 37° extracts showed a much lower misincorporation of isoleucine into polyphenylalanine in the poly U system. In addition, the Mg⁺⁺ concentration required for optimal translational activity was higher for the 37° than for the 30° extracts. Extracts from a culture grown in L medium at 37° or from a tif ^-/F tif ⁺ merodiploid grown at 37° with adenine behaved similarly to that from the 30° culture when poly U was used as messenger RNA. It is suggested that the tif ⁺ gene product may play a regulatory role in ribosomal function and the pleiotropic nature of the tif-1 mutation could be due to impairment of translational activity augmented by elevated temperature or by adenine.     

Response characteristics of a cold receptor in the stick insectCarausius morosus

Summary The cold cell in the easily identified mound-shaped sensillum on the 12th segment ofCarausius morosus' antennae responds to downward temperature (T) steps from about 15 °C with a sharp rise in impulse frequency (F). Responses to similar steps from higher initial temperatures are smaller (Figs. 1, 3, 4). As initialT increases from 16 °C to 31 °C, differential sensitivity to downward steps falls off by a factor of 27: to yield an average increase inF of 1 imp/s, steps down from 31 °C must increase by 1.7 °C; steps down from 16 °C, by only 0.06 °C (Fig. 5). Resolving power forT-steps at mid-range initial temperatures is about 0.7 °C, i.e. the probability that a single cold cell at average differential sensitivity will correctly discriminate between twoT steps 0.7 °C apart is 90% when the cell is presented with each step just once.The same cold cell also displays a clear dependence on steadyt between 14 °C and 24 °C (Figs. 7, 8). The static discharge rate of a single cell at average differential sensitivity has a resolving power of about 0.9 °C for steadyT. — The static discharge is not affected by the amount of water vapor in the stimulating air (Fig. 9).Abbreviations F impulse frequency in impulses per second (imp/s) - Pw partial pressure of water vapor in torr - r correlation coefficient - T temperature in °C - T step change inT     

High resolution 13C NMR spectra on oriented lipid bilayers: from quantifying the various sources of line broadening to performing 2D experiments with 0.2-0.3 ppm resolution in the carbon dimension

¹³C NMR spectra routinely performed on oriented lipid bilayers display linewidth of 1–2 ppm, although T₂ measurements indicate that 0.1–0.2 ppm could be obtained. We have prepared a DMPC – ¹³C₄-cholesterol (7/3) sample, and oriented the lipid bilayers between glass plates so that the bilayer normal makes an angle of 90° (or of the magic angle) with B₀. We have measured T₂s, CSAs, and linewidths for the choline ¹³C--methyl, the cholesterol-C₄ carbons and the lipid head group phosphorus, at both angles and 313 K. The magnetic field distribution within the sample was calculated using the surface current formalism. The line shapes were simulated as a function of B₀ field inhomogeneities and sample mosaic spread. Both effects contribute to the experimental linewidth. Using three signals of different CSA, we have quantified both contributions and measured the mosaic spread accurately. Direct shimming on a sample signal is essential to obtain sharp resonances and ¹³C labelled choline methyl resonance of DMPC is a good candidate for this task. After optimisation of the important parameters (shimming on the choline resonance, mosaic spread of ±0.30° ), ¹³C linewidth of 0.2–0.3 ppm have been obtained. This newly achieved resolution on bilayers oriented at 90°, has allowed to perform two 2D experiments, with a good sensitivity: 2D PELF (correlation of carbon chemical shifts and C-H dipolar couplings) and 2D D-resolved experiment (correlation of carbon chemical shifts and C-C dipolar couplings). A C-C dipolar coupling of 35 ± 2 Hz between the choline methyl carbons was determined.     

Genetically polymorphic α-l-fucosidase (FUCA1) isozymes detected in blood plasma

The main isozyme patterns of desialylated blood plasma or serum -l-fucosidase (FUCA) were found to be almost identical to those of semen, urine, placental extracts, and leukocyte lysates, when detected by polyacrylamide gel isoelectric focusing, and activity staining using the fluorogenic substrate 4-methylumbelliferyl--l-fucopyranoside. Three phenotypes (1, 2-1, and 2) determined from plasma samples were identical to the phenotypes from urine and leukocyte lysates from the same individuals. A population study of plasma samples collected from 485 Japanese individuals indicated that the frequencies of the FUCA11 ^* and FUCA12 ^* alleles were 0.7505 and 0.2495, respectively. The mean plasma enzyme activities (+SD) of the three phenotypes were 318.8 ± 116.7 nmol/ml per h for type 1, 268.0 ± 108.3 nmol/ml per h for type 2-1, and 233.2 ± 84.4 nmol/ml per h for type 2. The mean activities of types 1 and 2 suggest that, on average, the FUCA11 ^* gene product in plasma has about 1.4 times the activity of FUCA12 ^*.     

Flight of the honey bee

Summary Using manometric and gas analytical methods oxygen consumption , carbon dioxide production , respiratory quotientRQ, (Fig. 1A-C) and thorax surface temperature difference T _ts (Fig. 3) were determined in single bees. The animals were either sitting in respiratory chambers or were suspended by the scutum, in which case they were resting, walking (turning a small polystyrene ball) or flying in a closed miniature wind tunnel.During resting (sitting in Warburg vessels) at an ambient temperatureT _a=10°C,RQ was 1.01±0.2 (n=905) with variations due to method (Fig. 1D, E).RQ values during walking were determined in single cases. In no case were they significantly different from 1.00. After the first 10 min of flight meanRQ was 1.00±0.04. It was significantly smaller than 1.00 (RQ=0.97) only during the last 5% of long-time flights (mean flight duration 58.8±28.8 min). With the exception of near-exhaustion conditions no signs of fuels other than carbohydrates were found.Metabolic rateP _m was 19.71±21.38 mW g^–1 during resting at 20°CT _a30°C indicating that many resting bees actively thermoregulate at higherT _a. After excluding bees which were actively thermoregulating, by an approximationP _m was 5.65±2.44 mW g^–1 at 20°CT _a30°C. True resting metabolic rate for sitting bees atT _a=10°C was 1.31±0.53 mW g^–1 (Fig. 2A, B).A significant negative correlation was found between relative (specific) oxygen consumption rel and body massM _b at 85 mgM _b150 mg.At 0°CT _ts16.5°C a significant (-0.01) positive correlation was found between and T _ts in single resting bees: T _Ts+0.099, or betweenP _m and T _ts:P _m=1.343 T _ts+0.581 (Fig. 3D) in ml h^–1,P _m in mW,T in °C).During walking (duration 13.15±5.71 min,n=13) at 12.5°CT _a21°C a stable T _ts of 11.41±3.37°C, corresponding to 167 mW g^–1, was reached for 80 to 90% of the walking time (Fig. 4B).During wind tunnel flights of tethered animals the minimal metabolic power measured in exhaustion experiments was 240 mW g^–1. Calculation of factors of increase inP _m is of limited value in poikilotherms, in which true resting conditions are not exactly defined.     

Thermal instability of red blood cell membrane bilayers: Temperature dependence of hemolysis

Summary Rates of human red blood cell hemolysis were measured as a function of temperature. Three distinct temperature intervals for hemolysis were noted: a) At temperatures equal to or less than 37°C no hemolysis was observed for the duration of the incubation (30 hr). b) For temperatures exceeding 45°C hemolysis rates are rapid and are accompanied by gross changes in cellular morphology. The activation energy for hemolysis is 80 kcal/mole; this value is characteristic of protein denaturation and enzyme inactivation suggesting that these processes contribute to hemolysis at these high temperatures. c) Between 38 and 45°C the energy of activation is 29 kcal/mole, indicating that a fundamentally different process than protein inactivation is responsible for hemolysis at these relatively low temperatures. A mechanism based on the concept of the critical bilayer assembly temperature of cell membranes (N.L. Gershfeld,Biophys. J. 50:457–461, 1986) accounts for hemolysis at these relatively mild temperatures: The unilamellar state of the membrane is stable at 37°C, but is transformed to a multibilayer when the temperature is raised; hemolysis results because formation of the multibilayer requires exposing lipid-free areas of the erythrocyte surface. An analysis of the activation energy for hemolysis is presented that is consistent with the proposed unilamellar-multibilayer transformation.     

A comparison of proline, thiol levels and GAPDH activity in cyanobacteria of different origins facing temperature-stress

Three cyanobacterial strains originating from different habitats were subjected to temperature shift exposures and monitored for levels of proline, thiol and activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Thermophile Mastigocladus laminosus (growth optimum, 40 °C), raised the proline level 4.2-fold at low temperature (20 °C), for the psychrophile Nostoc 593 (growth optimum, 20 °C), it was raised 8-fold at 40 °C while in the mesophile Nostoc muscorum (growth optimum, 30 °C), the imino acid level increased 2.3-fold during temperature shiftdown to 20 °C or 3.5-fold in sets facing shiftup (40 °C). Alterations in thiol levels in the above strains were in line with proline. It is suggested that such fluctuations reflect metabolic shifts as a response to stress. Interestingly, GAPDH activity was maximum at the respective growth temperature optimum of M. laminosus (122 nmol NADPH oxidized min ^–1 mg ^–1 protein) and Nostoc 593 (141 nmol NADPH oxidized min ^–1 mg ^–1 protein) while in N. muscorum, it increased at 40 °C (101 nmol NADPH oxidized min ^–1 mg ^–1 protein) and to 93.3 nmol NADPH oxidized min ^–1 mg ^–1 protein (20 °C) relative to 86 nmol NADPH oxidized min ^–1 mg ^–1 protein at 30 °C. It seems that extremophiles maintain the GAPDH activity/level during growth at their respective temperatures optimal while the mesophile increases it in order to cope up with temperature-stress.     

Cold tolerance of Littorinidae from southern Africa: intertidal snails are not constrained to freeze tolerance

All intertidal gastropods for which cold tolerance strategies have been assessed have been shown to be freeze tolerant. Thus, freeze tolerance is considered an adaptation to the intertidal environment. We investigated the cold tolerance strategies of three species of subtropical and temperate snails (Gastropoda: Littorinidae) to determine whether this group is phylogenetically constrained to freeze tolerance. We exposed dry acclimated and wet rehydrated snails to low temperatures to determine temperature of crystallisation (Tc), lower lethal temperature and LT₅₀ and to examine the relationship between ice formation and mortality. Tc was lowest in dry Afrolittorina knysnaensis (–13.6±0.4 °C), followed by dry Echinolittorina natalensis (–10.9±0.2 °C) and wet A. knysnaensis (–10.2±0.2 °C). The Tc of both A. knysnaensis and E. natalensis increased with rehydration, whereas Tc of dry and wet Afrolittorina africana did not differ (–9.6±0.2 and –9.0±0.2 °C respectively). Wet snails of all species exhibited no or low survival of inoculative freezing, whereas dry individuals of A. knysnaensis could survive subzero temperatures above –8 °C when freezing was inoculated with ice. In the absence of external ice, Afrolittorina knysnaensis employs a freeze-avoidance strategy of cold tolerance, the first time this has been reported for an intertidal snail, indicating that there is no family-level phylogenetic constraint to freeze tolerance. Echinolittorina natalensis and A. africana both showed pre-freeze mortality and survival of some internal ice formation, but were not cold hardy in any strict sense.     

Brain temperature in pigeons: Effects of anterior respiratory bypass

Summary During heat stress in domestic pigeons (Columba livia, mean mass 0.43 kg) brain temperature (T _B) varied in parallel with colonic temperature (T _c). The difference between these (T _C–T _B=T) averaged 0.7°C and was not significantly altered when the animal breathed through a trachael cannula bypassing the buccopharyngeal cavity. When we sealed the nares and beak in bypass animals, T was significantly reduced but was nevertheless maintained at 0.4°C. When the eyes were sealed as well, however, T was reversed, amounting to –0.4°C. Conversely, with eyes sealed but beak and nares open, T was indistinguishable from that in controls. These results suggest a role for the cornea in evaporative cooling, at least when respiratory evaporation is impaired, and are consistent with the hypothesis that buccopharyngeal and corneal evaporation are coupled to brain cooling. The probable mechanism for this coupling is the flow of venous blood from evaporative surfaces through theretia mirabilia in the temporal areas. Here heat is transferred from the warmer arterial blood flowing through theretia toward the brain to the centrally flowing, cooler venous blood.     

Selectively 13C-enriched DNA: Evidence from 13C1′ relaxation rate measurements of an internal dynamics sequence effect in the lac operator

Summary In order to study some internal dynamic processes of the lac operator sequence, the ¹³C-labeled duplex 5d(C₀G₁C₂T₃C₄A₅C₆A₇A₈T₉T₁₀) · d(A₁₀A₉T₈T₇G₆T₅G₄A₃G₂C₁G₀)3 was used. The spreading of both the H1 and C1 resonances brought about an excellent dispersion of the ¹H1-¹³C1 correlations. The spinlattice relaxation parameters R(C_z), R(C_x,y) and R(H_zC_z) were measured for each residue of the two complementary strands, except for the 3-terminal residues which were not labeled. Variation of the relaxation rates was found along the sequence. These data were analyzed in the context of the model-free formalism proposed by Lipari and Szabo [(1982) J. Am. Chem. Soc., 104, 4546–4570] and extended to three parameters by Clore et al. [(1990) Biochemistry, 29, 7387–7401; and (1990) J. Am. Chem. Soc., 112, 4989–4991]. A careful analysis using a least-squares program showed that our data must be interpreted in terms of a three-parameter spectral density function. With this approach, the global correlation time was found to be the same for each residue. All the C1-H1 fragments exhibited both slow (_s = 1.5) and fast (_f = 20 ps) restricted libration motions (S _inf2 ^sups =0.74 to 1.0 and S _inf2 ^supf =0.52 to 0.96). Relaxation processes were described as governed by the motion of the sugar relative to the base and in terms of bending of the whole duplex. The possible role played by the special structure of the AATT sequence is discussed. No evident correlation was found between the amplitude motions of the complementary residues. The 5-terminal residues showed large internal motions (S²=0.5), which describe the fraying of the double helix. Global examination of the microdynamical parameters S _inf2 ^supf and S _inf2 ^sups along the nucleotide sequence showed that the adenine residues exhibit more restricted fast internal motions (S _inf2 ^supf =0.88 to 0.96) than the others, whereas the measured relaxation rates of the four nucleosides in solution were mainly of dipolar origin. Moreover, the fit of both R(C_z) and R(HzC_z) experimental relaxation rates using an only global correlation time for all the residues, gave evidence of a supplementary relaxation pathway affecting R(C_x,y) for the purine residues in the (53) G₄A₃ and A₁₀A₉T₈T₇ sequences. This relaxation process was analyzed in terms of exchange stemming from motions of the sugar around the glycosidic bond on the millisecond time scale. It should be pointed out that these residues gave evidence of close contacts with the protein in the complex with the lac operator [Boelens et al. (1987) J. Mol. Biol., 193, 213–216] and that these motions could be implied in the lac-operator-lac-repressor recognition process.     

Ca2+-Dependent and Ca2+-Independent [3H]GABA Release from Rat Brain Synaptosomes: the Effect of Temperature

The main inhibitory neurotransmitter GABA in the mammalian brain is distributed in the nerve terminals between two pools, vesicular (synaptic vesicles) and cytosolic. GABA is released from these pools by different mechanisms; there are calcium-activated exocytotic release and calcium-independent sodium-dependent release from the cytosolic pool (resulting from the membrane GABA transporter reversal). We investigated the influence of temperature on [³H]GABA release from rat brain synaptosomes, which was induced by stimulation of both these processes. In addition, we used -latrotoxin as a stimulant of [³H]GABA release. Synaptosomes from the rat brain were used in the experiments. 4-Aminopyridine (4-AP) and high [KCl] were applied to stimulate calcium-activated and calcium-independent [³H]GABA release, respectively. 4-AP-evoked [³H]GABA release was of the same intensity at 37 and 25°C (10.1 ± 1.2 and 10.1 ± 0.8% of total [³H]GABA incorporated into the synaptosomes, respectively). The effect of 4-AP on the ⁴⁵Ca²⁺ influx into synaptosomes was also temperature-independent: 0.775 ± 0.075 and 0.725 ± 0.100 nmol/min/mg of protein at 37 and 25°C, respectively. A drop in the effect of 4-AP was observed only at 15°C. When synaptosomes were depolarized with 50 mM KCl, a temperature decrease from 37°C to 25°C resulted in a twofold drop in the [³H]GABA release, from 20.5 ± 1.4 to 10.3 ± 0.7%; at 15°C [³H]GABA release dropped to less than one-third of the norm (6.0 ± 0.5%). -Latrotoxin-stimulated [³H]GABA release was diminished from 32.5 ± 2.5 at 37°C to 17.2 ± 1.3 at 25°C and 5.9 ± 0.4% at 15°C and was not affected by the presence or absence of calcium in the medium. It seems likely that the observed effect of temperature can be interpreted as based on the temperature dependence of the -latrotoxin insertion into the membrane. It is suggested that the pattern of the temperature sensitivity of GABA release from the synaptosomes can be used as a criterion for identification of the mode of neurotransmitter release.     

Coliphage 434 tofprotein: NH2-terminal amino acid sequence and kinetic and equilibrium measurements of DNA binding

Summary Coliphage 434 tof protein was purified to a substantially pure state from imm ⁴³⁴ cI dv carrier cells. The minimum molecular weight is 7,500±500 as estimated by polyacrylamide gel electrophoresis. The amino acid sequence of the nine NH₂-terminal residues was determined, by manual Edman degradation of the intact protein, to be Met-Gln-Thr-Leu-Ser-Glu-Arg-Leu-(Lys)-.The purified protein at low concentrations binds specifically to imm ⁴³⁴dv DNA and at high concentrations also binds to imm ²¹dv and dv DNA. The curve of the specific binding is of Michaelis type, while that of the nonspecific binding is sigmoidal. The specific binding does not show marked temperature dependency at 4°–37°C. We have analyzed the equilibrium and kinetic data of specific binding. The equilibrium dissociation constant is 1.9x10^-11M at 0°C. The association rate constant and the dissociation rate constant are 1.1–2.9x10⁸M^-1s^-1 and 2.7x10^-3s^-1, respectively, at 0°C. The half life of the tof protein-operator DNA complex is 260s. These results suggest that the tof protein-operator interaction is much weaker than the interaction between the cI repressor and the operator reported by other workers.     

Flight of the honey bee VI: energetics of wind tunnel exhaustion flights at defined fuel content,speed adaptation and aerodynamics

To gain information on extended flight energetics, quasi-natural flight conditions imitating steady horizontal flight were set by combining the tetheredflight wind-tunnel method with the exhaustion-flight method. The bees were suspended from a two-component aerodynamic balance at different, near optimum body angle of attack and were allowed to choose their own speed: their body mass and body weight was determined before and after a flight; their speed, lift, wingbeat frequency and total flight time were measured throughout a flight. These values were used to determine thrust, resultant aerodynamic force (magnitude and tilting angle), Reynolds number, total flight distance and total flight impulse. Flights in which lift was body weight were mostly obtained. Bees, flown to complete exhausion, were refed with 5, 10, 15 or 20 l of a 1.28-mol·l^-1 glucose solution (energy content w=18.5, 37.0, 55.5 or 74.0 J) and again flown to complete exhaustion at an ambient temperature of 25±1.5°C by a flight of known duration such that the calculation of absolute and relative metabolic power was possible. Mean body mass after exhaustion was 76.49±3.52 mg. During long term flights of 7.47–31.30 min similar changes in flight velocity, lift, thrust, aerodynamic force, wingbeat frequency and tilting angle took place, independent of the volume of feeding solution. After increasing rapidly within 15 s a more or less steady phase of 60–80% of total flight time, showing only a slight decrease, was followed by a steeper, more irregular decrease, finally reaching 0 within 20–30 s. In steady phases lift was nearly equal to resultant aerodynamic force; tilting angle was 79.8±4.0°, thrust to lift radio did not vary, thrust was 18.0±7.4% of lift, lift was somewhat higher/equal/lower than body mass in 61.3%, 16.1%, 22.6% of all totally analysable flights (n=31). The following parameters were varied as functions of volume of feeding solution (5–20 l in steps of 5 l) and energy content. (18.5–74.0 J in steps of 18.5 J): total flight time, velocity, total flight distance, mean lift, thrust, mean resultant aerodynamic force, tilting angle, total flight impulse, wingbeat frequency, metabolic power and metabolic power related to body mass, the latter related to empty, full and mean (=100 mg) body mass. The following positive correlations were found: L=1.069·10^-9 f ^2.538; R=1.629·10^-9 f ^2.464; P _m=7.079·10^-8 f ^2.456; P _m=0.008v+0.008; P _m=18.996L+0.022; P _m=19.782R+0.021; P _m=82.143T+0.028; P _m=1.245·bm _f ^1.424 ; P _{mrel e}=6.471·bm _f ^1.040 ; =83.248+0.385. The following negative correlations were found: V=3.939–0.032; T=1.324·10^-4–0.038·10^-4. Statistically significant correlations were not found in T(f), L(), R(), f(), P _m(bm _e), P _{m rel e}(bm _e), P _{m rel f}(bm _e), P _{m rel f}(bm _f).Abbreviations A(m²) frontal area - bl(m) body length - bm(mg) body mass - c(mol·1^-1) glucose concentration of feeding solution - c _D (dimensionless) drag coefficient, related to A - D(N) drag - F _w(N) body weight - F _wp weight of paper fragment lost at flight start - f wingbeat frequency (s^-1) - g(=9.81 m·s^-2) gravitational acceleration - I(Ns)=R(t) dt total impulse of a flight - L(N) lift vertical sustaining force component - P _m(J·s^-1=W) metabolic power - P_{m ret} (W·g^-1) metabolic power, related to body mass - R(N) resultant aerodynamic force - Re v·bl·v ^-1 (dimensionless) Reynolds number, related to body length - s(m) v(t) dt virtual flight distance of a flight - s(km) total virtual flight distance - T (N) thrust horizontal force component of horizontal flight - T _a (°C) ambient temperature - t(s) time - t _tot (s or min) total flight time - v(m·s^-1) flight velocity - v(l) volume of feeding solution - W (J) energy and energy content of V - ( °) body angle of attack between body longitudinal axis and flow direction - ( °) tilting angle ( 90°) between R and the horizont in horizontal flight v(=1.53·10^-5m²·s^-1 for air at 25°) kinematic viscosity - (=1.2 kg·m^-3 at 25°C) air density     

Responsiveness of cardiac Na+ channels to antiarrhythmic drugs: The role of inactivation

Summary Elementary Na⁺ currents were recorded at 9°C in inside-out patches from cultured neonatal rat heart myocytes. In characterizing the sensitivity of cooled, slowly inactivating cardiac Na⁺ channels to several antiarrhythmic drugs including propafenone, lidocaine and quinidine, the study aimed to define the role of Na⁺ inactivation for open channel blockade.In concentrations (1–10 mol/liter) effective to depressNP _o significantly, propafenone completely failed to influence the open state of slowly inactivating Na⁺ channels. With 1 mol/liter, _open changed insignificantly to 96±7% of the control. Even a small number of ultralong openings of 6 msec or longer exceeding _open of the whole ensemble several-fold and attaining _open (at –45 mV) in cooled, (-)-DPI-modified, noninactivating Na⁺ channels proved to be drug resistant and could not be flicker-blocked by 10 mol/liter propafenone. The same drug concentration induced in(-)-DPI-modified Na⁺ channels a discrete block with association and dissociation rate constants of 16.1 ± 5.3 × 10⁶ mol^–1 sec^–1 and 675 ± 25 sec^–1, respectively. Quinidine, known to have a considerable affinity for activated Na⁺ channels, in lower concentrations (5 mol/liter) left _open unchanged or reduced, in higher concentrations (10 mol/liter) _open only slightly to 81% of the predrug value whereasNP _o declined to 30%, but repetitive blocking events during the conducting state could never be observed. Basically the same drug resistance of the open state was seen in cardiac Na⁺ channels whose open-state kinetics had been modulated by the cytoplasmic presence of F^– ions. But in this case, propafenone reduced reopening and selectively abolished a long-lasting open state. This drug action is unlikely related to the inhibitory effect onNP _o since hyperpolarization and the accompanying block attenuation did not restore the channel kinetics. It is concluded that cardiac Na⁺ channels cannot be flicker-blocked by antiarrhythmic drugs unless Na⁺ inactivation is removed.     

Stimulus-evoked slow potential shifts and changes in [K+]0 of the frog optic tectum

Summary In 17 frogs (Rana esculenta var ridibunda) immobilised with succinyl choline the optic tectal surface was stimulated by trains of electrical pulses or by a flash to the contralateral eye. Sustained potential shifts (SPSs) and changes in extracellular potassium concentration ( [K⁺]₀) were simultaneously recorded.In response to electrical stimulation SPSs of maximal amplitudes (1.19±0.1 mV) were recorded between 50 and 200 m in depth and maximal [K⁺]₀ (0.69 ±0.08 mM) between 100 and 350 m. The changes of SPS and [K⁺]₀ showed a close similarity in experiments with changes in voltage, pulse duration and frequency of stimuli within a train. The induced SPS had a duration of 28±1.54 s, the [K⁺]₀ of 32±1.23 s.The flash stimulus induced an SPS and [K⁺]₀ of maximal amplitudes between 50 and 200 m in depth with values of 0.57±0.1 mV and 0.29±0.03 mM respectively. An additional wave with a latency of ca 1 s and a duration of ca 3 s arose on the background of the SPS to a flash stimulus, associated with an additional increase in [K⁺]₀.It is considered that the accumulation of K⁺ in extra-cellular space, with neuronal activity, results in depolarization of radial processes of ependymal glia. This is reflected in the neuropil of the upper layers of the optic tectum as an SPS.We would like to dedicate this article to the memory of Alexander Roitbak who died as a result of a tragic accident while this paper was in press. He will be remembered fondly especially for his contributions to understanding of the functions of Neuroglia. E.V.O., P.R.L., T.A.R.