Phosphorylation of chicken gizzard myosin: Myosin filament hypothesis of calcium regulation

Myosin from chicken gizzard smooth muscle was found to be characteristically different from rabbit skeletal striated myosin: i) ATP induced a profound change in the conformation of gizzard myosin molecules. ii) ATP also induced disassembling of gizzard myosin filaments. iii) Enzymic phosphorylation of gizzard myosin light chains rendered both the myosin conformation and the myosin filaments resistant to the actions of ATP. iv) Very high concentrations of magnesium were required for formation of the ATP-resistant filaments as well as for superprecipitation (a model contraction) of actomyosin suspensions. v) ITP was a very poor substrate for MLCK, and was accordingly incapable of inducing “Ca-tension” in glycerinated fibers of gizzard muscle, but it did induce “Mg-tension.” Primarily from these findings, it was proposed that tje mechanism of gizzard muscle contraction involves ATP-induced changes in the morphology of myosin filaments which are reversibly altered by enzymic phosphorylation and dephosphorylation of myosin light chains in the presence of relatively high concentrations of magnesium.     

Effect of diet on the weight of brown adipose tissue in rodents

Interscapular brown adipose tissue (BAT) weighed more in rats given access to a solution of sucrose in addition to a nutritionally complete basal diet than in rats eating only a basal diet. This incremental effect of drinking sucrose solution occurs across a variety of dietary conditions. In the first experiment, rats were fed diets containing either 9%, 18%, 27% or 36% casein. Rats given access to a sucrose solution had significantly larger brown fat pads than controls when the diets contained 9 or 18% casein, but not when diets contained either 27% or 36% casein. The second experiment examined the weight of brown adipose tissue as a function of the type of protein and the percentage of fat in the diet. Animals given a sucrose solution had significantly more BAT than animals not given sucrose. Neither the type of protein (casein or soy protein) nor the percentage of fat (14.5% or 36.4%) in the diet influenced the weight of BAT. Animals given access to either a sucrose solution or a glucose solution had significantly heavier BAT than animals given access to a fructose solution, granulated sucrose or water.     

The effect of salts on catecholamine fluxes and adenosine triphosphatase activity in storage vesicles from the adrenal medulla

1. Influx and efflux of catecholamine and adenosine triphosphatase activity in storage vesicles from the adrenal medulla were studied with dl-[(14)C]adrenaline in different media. 2. The lowest values for flux and adenosine triphosphatase activity were observed in sucrose media in which an ATP-dependent influx of catecholamine compensated for an efflux of the same magnitude. Efflux in the presence or absence of ATP was similar. 3. In media containing sodium succinate or glutarate adenosine triphosphatase activity was higher and the ATP-dependent influx of catecholamine was about twice that observed in iso-osmotic sucrose medium. In the presence of ATP influx and efflux of catecholamine were balanced; in its absence there was a net release of catecholamine, since efflux was more than twice the influx. Efflux in the presence or absence of ATP was similar. 4. In media containing sodium or potassium chloride and in the presence of ATP influx and adenosine triphosphatase activity were further enhanced, but in the absence of ATP there was no further increase in influx, since catecholamine was released with or without ATP at the same rate. Efflux was therefore twice as high in the presence of ATP as in its absence. 5. Sodium nitrate suppressed the ATP-dependent influx nearly completely, but caused a greatly enhanced efflux, which was twice as high in the presence of ATP as in its absence. 6. The extinction of vesicular suspensions remained unchanged in the presence of ATP under conditions where the catecholamine efflux was balanced by the influx. Under conditions where the efflux was not compensated by influx, the extinction of the suspensions decreased in the presence of ATP more than in its absence.     

Formation in vitro of matrix-like aggregates of chromogranins and phospholipids derived from chromaffin granules of the bovine adrenal medulla

Matrix-like particles were formed in sonicated suspensions of soluble chromogranins and phospholipids extracted from the isolated membrane phase of chromaffin granules. The artificial particles which sedimented into 0.4–0.6 M sucrose layers during isopycnic centrifugation, consisted of liposomal vesicles and granular aggregates indicative of lamellar and possibly hexagonal organization patterns of phospholipids and the aqueous phase containing the soluble chromogranins. The ultrastructure of the granular aggregates was strikingly similar to the matrix phase of the intact chromaffin granule.The dopamine-β-hydroxylase activity (EC 1.14.2.1) of the soluble chromogranin preparation was potentiated by the addition of the phospholipids and further enhanced by the sonication procedure. The enzymic activity was highest in the 0.4–0.6 M sucrose layers, rich in the artificial matrix-like particles.The artificially formed particles incorporated ATP by further sonication and the presence of ATP in the particle fractions did not inhibit the specific dopamine-β-hydroxylase activity, even at ratios of 4 and 2 μmoles ATP/mg protein in the 0.4 and 0.6 M sucrose layers, respectively. Noradrenaline was incorporated into the particle fractions in presence of ATP. However, the presence of noradrenaline above 3·10^?6 M resulted in a 50–75% inhibition of the dopamine-β-hydroxylase activity of the artificial matrices.     

Antiplatelet effects of chelerythrine chloride isolated from Zanthoxylum simulans

Chelerythrine chloride is an antiplatelet agent isolated from Zanthoxylum simulans. Aggregation and ATP release of washed rabbit platelets caused by ADP, arachidonic acid, PAF, collagen, ionophore A23187 and thrombin were inhibited by chelerythrine chloride. Less inhibition was observed in platelet-rich plasma. The thromboxane B2 formation of washed platelets caused by arachidonic acid, collagen, ionophore A23187 and thrombin was decreased by chelerythrine chloride. Phosphoinositides breakdown caused by collagen and PAF was completely inhibited by chelerythrine chloride, while that of thrombin was only partially suppressed. Chelerythrine chloride inhibited the intracellular calcium increase caused by arachidonic acid, PAF, collagen and thrombin in quin-2/AM-loaded platelets. The cyclic AMP level of washed platelets did not elevated by chelerythrine chloride. The antiplatelet effect of chelerythrine chloride was not dependent on the incubation time and the aggregability of platelets inhibited by chelerythrine chloride was easily recovered after sedimenting the platelets by centrifugation and then the platelet pellets were resuspended. Chelerythrine chloride did not cause any platelet lysis, since lactate dehydrogenase activity was not found in the supernatant. These data indicate that the inhibitory effect of chelerythrine chloride on rabbit platelet aggregation and release reaction is due to the inhibition on thromboxane formation and phosphoinositides breakdown.     

Effect of salt concentration and unfrozen water fraction on the viability of slowly frozen ram spermatozoa

Ram spermatozoa were subjected to a slow rate of freezing (1 degree C/min) in various glycerol-NaCl-water solutions of known composition such that the molal concentration of NaCl (ms) and the unfrozen fraction of water (U) could be calculated at subzero temperatures from the relevant phase diagram. Sperm motility was reduced as ms increased and U correspondingly decreased with temperature. However, by freezing spermatozoa in solutions of differing initial tonicities, but with a constant weight ratio of glycerol: salt, to various subzero temperatures, the effects of ms could be separated from those of U. Motility was found to decrease dramatically at values of U less than 0.07 regardless of ms but, at higher values of U, maximum motility was dependent on the final salt concentration in that fraction, being reduced as the osmolality increased. Sperm cell concentration had no apparent effect on the influence of ms or U on viability in the range studied (3-12 x 10(8) spermatozoa/ml). In order to account for these observations, the effects of osmotic stress on spermatozoa were investigated. When subjected to sudden changes in osmolality of the suspending medium by increasing NaCl or sucrose concentration at room temperature, spermatozoa showed a decreased motility with increasing osmolality. Since no improvement in motility was found on returning the cells to isosmolar conditions cell damage appeared to be irreversible. Furthermore, when placed in solutions of increasing hypotonicity the number of swollen spermatozoa with looped tails increased with increasing hypotonicity. Since the drop in motility seen at low values of U corresponded to those spermatozoa exposed to a hypotonic starting solution, it is suggested that a hypotonic stress followed by a hypertonic stress during freezing and thawing may account for the profound loss of motility in these samples, while a hypertonic stress may account for the strong effect of ms seen at higher values of U.     

Comparison of the instrumental reinforcer devaluation effect in two strains of rats (Wistar and Lister)

In this experiment, the effect of the reinforcer devaluation upon instrumental performance was analysed in two strains of rats (Wistar and Lister): Food deprived rats were trained to press a lever for sucrose pellets in a single session. Immediately after the fulfilment of this session, half of the Wistar and Lister rats received an injection of lithium chloride (LiCl), while the remaining animals were injected with a sodium chloride (NaCl) solution. A subsequent extinction test showed that the subjects who had received immediate LiCl did not press the lever as often as those injected with NaCl. No differences in response suppression were found between the two strains of rats. These results also show that a single devaluation experience is sufficient for an impact on instrumental performance.     

THE EMERGENCE OF LARVAE FROM CYSTS OF THE BEET EELWORM, HETERODERA SCHACHTII SCHMIDT, IN AQUEOUS SOLUTIONS OF ORGANIC AND INORGANIC SUBSTANCES

The rate of larval emergence from cysts of the beet eelworm in a variety of aqueous solutions containing organic and inorganic substances is significantly higher than the emergence rate in water. It is suggested that differences between larval emergence rates in monoamino-monocarboxylic amino-acids may be related to the lipid solubility of these substances and their ability to penetrate the egg membranes. The larval emergence rate in fructose, glucose, sucrose and maltose was significantly higher than that in water, but in raffinose, arabinose and xylose the rate of emergence was no higher than in water. A high rate of larval emergence occurred in sodium chloride, potassium chloride and mercuric chloride, but not in magnesium chloride or calcium chloride. Experiments with several other organic solutions are described. There is an optimum concentration for larval emergence in beet diffusate. The osmotic pressure of the diffusate when maximum emergence occurred was 0·48 atm. Measurements of shrinkage of unhatched larvae in various concentrations of urea, sodium chloride and sucrose showed that decreasing rates of emergence at higher concentrations may be due to changes in the unhatched larvae brought about: by an osmotic effect. High concentrations of beet diffusate may have a similar effect.     

Damage to Escherichia coli on Exposure to Moist Heat

The effects of temperatures of 50 to 60 C on suspensions of a strain of Escherichia coli are described. At these temperatures, the substances which leaked from the cells were determined as pentoses; the amount leaked over a 30-min period increased with increasing temperature. The leakage materials from suspensions heated in water, sodium chloride, sucrose, and sucrose plus Mg(++) were examined spectrophotometrically, and the ratios of the absorbances at 280 and 260 nm (the 280 to 260 nm ratio) were determined. It was not possible to determine protein by this method, as the ratio was always <0.7. When saline was the suspending medium, the ratio was lower than when water was used, and there was a greater leakage of 260 nm absorbing material. When suspended in sucrose or in sucrose plus Mg(++), penicillin-induced spheroplasts did not undergo lysis, but became less regular in shape, and there was an increase in the extinction at 500 nm. The overall effects of high temperatures on nonsporeforming bacterial cells are discussed; in view of the available evidence, it is concluded that ribonucleic acid degradation is an event which is probably closely related to thermally induced bacterial death.     

THE ELONGATION OF MYOFIBRILS FROM THE INDIRECT FLIGHT MUSCLE OF DROSOPHILA

Myofibrils which lengthen by several per cent in the presence of ATP and magnesium ions were prepared by teasing indirect flight muscle of Drosophila in solutions containing ethylenediaminetetraacetate. A study was made of the hydrogen ion, magnesium ion, ATP, and potassium chloride concentrations with which this effect could be observed. The lack of elongation with pyrophosphate and several nucleoside triphosphates suggests that the lengthening is ATP specific. A relaxing factor system comparable to that described for rabbit muscle was not demonstrable, as elongated fibrils did not shorten with calcium ions, carnosine, or digitonin.     

Inhibition of regeneration in the planarian Dugesia polychroa (Schmidt) by treatment with magnesium chloride: a morphological study of wound closure

At a concentration of 0.2% (21 m M) in culture water, magnesium chloride impaired muscle contraction and completely inhibited head regeneration in specimens of Dugesia polychroa cut prepharyngeally. The wound stayed open for nine days, with neoblasts accumulating beneath the wound without any signs of differentiation. Extremely delayed wound closure occurred by spreading epithelial cells, and was completed after 30 days in the magnesium chloride solution. Histological examination confirmed the absence of any regenerated head structures. Interestingly, the inhibitory effect was removed when such headless fragments were cut once more and kept in normal culture water: complete head regeneration then occurred at a normal rate. Among several possible explanations for the failure to regenerate, the following hypothesis is an attractive alternative: direct contact between parenchyma and epithelial cells during the period following injury seems to be an essential stimulus for the start of cell differentiation within the blastema, and the lack of such contact as a result of the drug action prevents normal regeneration. When the wound has eventually closed, a continuous basement membrane separates epithelium from parenchyma. Thus a direct contact between these tissues is never established. This revised version was published online in July 2006 with corrections to the Cover Date.     

Possible role of structural changes in ATP hydrolysis by subfragment-1 and myosin

Dependence of the rate of ATP hydrolysis with subfragment-I and temperature of SF-I, denaturation on the concentration of heavy water in solution was studied. The value of kinetic isotope effect V/Vx linearly increases with the rise of the volume portion of heavy water in solution and at X-1 it equals 1.9. The temperature of protein denaturacticn increases with X rise, the pattern of this relationship looking as an arched curve. The results differ from those earlier obtained on myosin which points to the absence of essential contribution of structural dynamic changes to enzymic hydrolysis of ATP by subfragment-I.     

Desiccation tolerance in bovine sperm: A study of the effect of intracellular sugars and the supplemental roles of an antioxidant and a chelator

Desiccation preservation holds promise as a simplified alternative to cryopreservation for the long term storage of cells. We report a study on the protective effects of intracellular and extracellular sugars during bovine sperm desiccation and the supplemental effects of the addition of an antioxidant (catalase) or a chelator (desferal). The goal of the study was to preserve mammalian sperm in a partially or completely desiccated state. Sperm loaded intracellularly with two different types of sugars, trehalose or sucrose, were dried with and without catalase and desferal and evaluated for motility and membrane integrity immediately after rehydration. Intracellular sugars were loaded using ATP induced poration. Drying was performed in desiccator boxes maintained at 11% relative humidity (RH). Results indicated that sperm exhibited improved desiccation tolerance if they were loaded with either intracellular trehalose or sucrose. Survival was further enhanced by the addition of 1 mM desferal to the desiccation buffer. Though sperm motility after drying to low dry basis water fractions (DBWF) did not show significant improvement under any of the tested conditions, there was an increase in the sperm membrane integrity that could be retained after partial desiccation through the use of intracellular sugars and desferal.     

Effects of litter size on sympathetic activity in young adult rats

Rearing animals in small litters induces a permanent increase in body weight and body fat. To determine whether changes in sympathoadrenal activity contribute to this effect, litter size was adjusted the day after birth and maintained until weaning at 21 days. Sympathetic nervous system (SNS) activity was measured in adult animals using [(3)H]norepinephrine ([(3)H]NE) turnover in peripheral tissues. Although litter size was without effect on [(3)H]NE turnover in chow-fed animals, acceleration of [(3)H]NE turnover by dietary sucrose was completely abolished in heart and attenuated in interscapular brown adipose tissue and kidney of rats reared in small litters. Body and epididymal fat-pad weights were heavier in rats reared in small litters; however, weight gain in response to dietary enrichment with sucrose did not differ as a function of litter size. Thus litter size alters dietary activation of the SNS, and this effect presumably reflects changes in central nervous system regulation.     

呼吸活性的下降可解释水分亏缺对银合欢根瘤固氮酶活性的抑制(英)

在温室条件下研究了水分亏缺对银合欢根瘤的水分关系、固氮酶活性（乙炔还原活性）、呼吸活性以及蔗糖代谢有关酶活性的影响。随着土壤含水量的下降,根瘤水势也相继下降。土壤干旱不但显著地抑制了根瘤乙炔还原的活性,而且对根瘤的呼吸活性、ATP的产生以及催化蔗糖降解的碱性转化酶和蔗糖合成酶的活性也具强烈的抑制作用。然而,根瘤可溶性总糖含量则不受土壤干旱的影响。用呼吸抑制剂DNP处理根瘤后,其固氮酶活性、呼吸活性及ATPI含量都受到极显著的抑制。这都表明,水分胁迫对根瘤呼吸活性及ATP产生的抑制可解释干旱条件下固氮酶活性的下降。     

An analysis of physiological states responsible for antero-posterior disintegration in Planaria dorotocephala

Summary Planaria were treated with equi-molal solutions of ammonium, potassium, sodium, magnesium, and calcium chlorides, made up in distilled water and the rates of cytolysis compared with cytolysis in distilled water. Potassium and ammonium accelerate cytolysis; some protection is afforded by sodium; still more by magnesium, and complete protection by calcium in the concentrations employed.In distilled water solutions of calcium chloride no cytolysis occurs in concentrations from M/500 to M/40,000; cytolysis is distinctly delayed in M/100,000. The protective action of M/1,000,000 is detectable.Potassium oxalate accelerates disintegration in hypotonic solutions.One per cent ethyl alcohol in distilled water causes cytolysis more rapidly than does distilled water alone, but in M/500 molal calcium chloride the alcohol solution is much less effective.Ringer's solution minus calcium affords no protection against death due to absence of calcium and death due to potassium oxalate but completely protects against cytolysis. Death in Ringer's solution minus calcium and in Ringer's solution with potassium oxalate occurs first in the anterior region and describes an antero-posterior gradient.Cytolysis in distilled water, in potassium oxalate solutions, in alcohol solutions, and in hypotonic calcium solutions of extreme dilution is initiated in the anterior end and describes an antero-posterior gradient within a zooid.Earlier work of the writer on the disintegrative action of lipoid solvents, heat, KNC, hyper- and hypotonic solutions is discussed. It is concluded that inPlanaria dorotocephala the antero-posterior gradient in cytolytic disintegration represents an antero-posterior differential in sensitivity to disturbance of the calcium-lipoid-water relation in the organism.     

Permeability of Rat Liver Microsomes to Sucrose and Carboxypoly-glucose in vitro

A study was made of the permeability of the microsomes to C¹⁴-sucrose and to C¹⁴-carboxypolyglucose, a branch-chained glucose polymer with a molecular weight of approximately 50,000. It was concluded that the microsomal membranes are permeable to sucrose on the basis of the following evidence: the volume of distribution of C¹⁴-sucrose was 84 per cent of the total microsomal pellet water; the sucrose unavailable volume, the per cent dry weights of the microsomal pellets, and the optical density of microsomal suspensions were independent of the concentration of sucrose in the suspending medium. It is suggested that the microsomal water which is unavailable to sucrose may be bound to protein and/or ribonucleic acid of the microsomes. The volume of distribution of C¹⁴-carboxypolyglucose was 44 per cent of the total pellet water, and it is considered that the microsomal membranes may be impermeable to this compound. Pretreatment with ribonuclease resulted in small increases in the volumes of distribution of both C¹⁴-sucrose and C¹⁴-carboxypolyglucose.     

Methanogenesis and ATP synthesis in a protoplast system of Methanobacterium thermoautotrophicum.

When Methanobacterium thermoautotrophicum cells were incubated in 50 mM potassium phosphate buffer (pH 7.0) containing 1 M sucrose and autolysate from Methanobacterium wolfei, they were transformed into protoplasts. The protoplasts, which possessed no cell wall, lysed in buffer without sucrose. Unlike whole cells, the protoplasts did not show convoluted internal membrane structures. The protoplasts produced methane from H2-CO2 (approximately 1 mumol min-1 mg of protein-1) at about 50% the rate obtained for whole cells, and methanogenesis was coupled with ATP synthesis. Addition of the protonophore 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF-6847) to protoplast suspensions resulted in a dissipation of the membrane potential (delta psi), and this was accompanied by a parallel decrease in the rates of ATP synthesis and methanogenesis. In this respect protoplasts differed from whole cells in which ATP synthesis and methanogenesis were virtually unaffected by the addition of the protonophore. It is concluded that the insensitivity of whole cells to protonophores could be due to internal membrane structures. Membrane preparations produced from lysis of protoplasts or by sonication of whole cells gave comparatively low rates of methanogenesis (methylcoenzyme M methylreductase activity, less than or equal to 100 nmol of CH4 min-1 mg of protein-1), and no coupling with ATP synthesis could be demonstrated.     

Trout sperm motility. The transient movement of trout sperm is related to changes in the concentration of ATP following the activation of the flagellar movement

For freshwater fish the motile period of sperm is extremely brief, even after a dilution in isotonic media. This result is in contrast to most other animals (ranging from invertebrates to mammals), in which sperm are generally motile for at least several hours. We have analyzed the reasons for the brevity of this movement by studying the relationships between the metabolism of trout sperm and the activation of their motility upon dilution. Sperm motility was not initiated when the dilution medium contained an elevated concentration of potassium (20-40 mM), but dilution in an isotonic solution of sodium chloride triggered an immediate activation of motility, and sperm swam vigorously. Motility of sperm decreased rapidly and 15 s after dilution sperm were moving slowly in small circles. Sperm became abruptly immotile at 20-30 s and flagella straightened. When millimolar concentrations of Ca2+ were also present in the dilution medium, movement did not stop abruptly, flagella kept beating and stopped only after 1-2 min. When sperm remained immotile they retained a high concentration of ATP. The activation of motility induced a rapid decrease of ATP. In the absence of calcium, and after the cessation of motility, ATP increased slowly back to its original concentration. In the presence of millimolar concentrations of calcium the concentration of ATP decreased to a very low level and remained low thereafter. The progressive decrease of the flagellar beat frequency, that had been observed during the period of trout sperm movement, might be related to the rapid exhaustion of intraflagellar ATP. Motility could be reinduced in sperm that had recovered high concentrations of ATP, demonstrating the functional integrity of the motile apparatus even after flagellar arrest. In conclusion we suggest that the maximum duration of trout sperm motility, at most 2 min (as a consequence of a depletion of ATP during the movement), is due to a low mitochondrial oxidative phosphorylation capacity.     

Properties of the high-affinity single-stranded DNA binding state of the Escherichia coli recA protein

The properties of the high-affinity single-stranded DNA (ssDNA) binding state of Escherichia coli recA protein have been studied. We find that all of the nucleoside triphosphates that are hydrolyzed by recA protein induce a high-affinity ssDNA binding state. The effect of ATP binding to recA protein was partially separated from the ATP hydrolytic event by substituting calcium chloride for magnesium chloride in the binding buffer. Under these conditions, the rate of ATP hydrolysis is greatly inhibited. ATP increases the affinity of recA protein for ssDNA in a concentration-dependent manner in the presence of both calcium and magnesium chloride with apparent Kd values of 375 and 500 microM ATP, respectively. Under nonhydrolytic conditions, the molar ratio of ATP to ADP has an effect on the recA protein ssDNA binding affinity. Over an ATP/ADP molar ratio of 2-3, the affinity of recA protein for ssDNA shifts cooperatively from a low-to a high-affinity state.