Steady-state Degree Research Articles

Modification of fos Proteins: Phosphorylation of c-fos, but Not v-fos, Is Stimulated by 12-Tetradecanoyl-Phorbol-13-Acetate and Serum

We have investigated the covalent modification of the proteins encoded by the murine fos proto-oncogene (c-fos) and that of the corresponding gene product of FBJ murine osteosarcoma virus (v-fos). Both proteins are posttranslationally processed in the cell, resulting in forms with lower electrophoretic mobilities than that of the initial translation product on sodium dodecyl sulfate-polyacrylamide gels. Treatment with alkaline phosphatase indicates that most, if not all, of this electrophoretic shift is due to phosphoesterification of both proteins. These phosphoryl groups stoichiometrically modify the v-fos and c-fos proteins on serine residues and turn over rapidly in vivo in the presence of protein kinase inhibitors (half-life, less than 15 min). Direct quantitative comparison of steady-state labeling studies with L-[35S]methionine and [32P]phosphate reveals that the c-fos protein is four- to fivefold more highly phosphorylated than the v-fos protein is. Comparison of tryptic fragments from [32P]phosphate-labeled proteins indicates that although the two proteins have several tryptic phosphopeptides in common, the c-fos protein contains unique major tryptic phosphopeptides that the v-fos protein lacks. These unique sites of c-fos phosphorylation have been tentatively localized to the carboxy-terminal 20 amino acid residues of the protein. Phosphorylation of the c-fos protein, but not the v-fos protein, can be stimulated at least fivefold in vivo by the addition of either 12-tetradecanoyl-phorbol-13-acetate or serum. This increase in the steady-state degree of phosphorylation of c-fos appears to be independent of protein kinase C since phosphorylation is Ca2+ and diacylglycerol independent. The possible role of phosphorylation of these proteins in cellular transformation is discussed.

Modification of fos proteins: phosphorylation of c-fos, but not v-fos, is stimulated by 12-tetradecanoyl-phorbol-13-acetate and serum.

We have investigated the covalent modification of the proteins encoded by the murine fos proto-oncogene (c-fos) and that of the corresponding gene product of FBJ murine osteosarcoma virus (v-fos). Both proteins are posttranslationally processed in the cell, resulting in forms with lower electrophoretic mobilities than that of the initial translation product on sodium dodecyl sulfate-polyacrylamide gels. Treatment with alkaline phosphatase indicates that most, if not all, of this electrophoretic shift is due to phosphoesterification of both proteins. These phosphoryl groups stoichiometrically modify the v-fos and c-fos proteins on serine residues and turn over rapidly in vivo in the presence of protein kinase inhibitors (half-life, less than 15 min). Direct quantitative comparison of steady-state labeling studies with L-[35S]methionine and [32P]phosphate reveals that the c-fos protein is four- to fivefold more highly phosphorylated than the v-fos protein is. Comparison of tryptic fragments from [32P]phosphate-labeled proteins indicates that although the two proteins have several tryptic phosphopeptides in common, the c-fos protein contains unique major tryptic phosphopeptides that the v-fos protein lacks. These unique sites of c-fos phosphorylation have been tentatively localized to the carboxy-terminal 20 amino acid residues of the protein. Phosphorylation of the c-fos protein, but not the v-fos protein, can be stimulated at least fivefold in vivo by the addition of either 12-tetradecanoyl-phorbol-13-acetate or serum. This increase in the steady-state degree of phosphorylation of c-fos appears to be independent of protein kinase C since phosphorylation is Ca2+ and diacylglycerol independent. The possible role of phosphorylation of these proteins in cellular transformation is discussed.

Methylation of calmodulin at carboxylic acid residues in erythrocytes. A non-regulatory covalent modification?

The physiological role of protein carboxy-group methylation reactions in human erythrocytes was studied with calmodulin as an endogenous methyl-group acceptor. The steady-state degree of calmodulin carboxy-group methylation is substoichiometric both in intact cells and in a lysed-cell system (about 0.0003 mol of methyl groups/mol of polypeptide). Purified erythrocyte calmodulin is a substrate for a partially purified erythrocyte carboxy-group methyltransferase and can be methylated to the extent of about 0.0007-0.001 mol of methyl groups/mol of polypeptide. This erythrocyte protein methyltransferase displays an apparent specificity for atypical racemized and/or isomerized D-aspartate and L-isoaspartate residues [McFadden & Clarke (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2460-2464; Murray & Clarke (1984) J. Biol. Chem. 259, 10722-10732]. Exposure of calmodulin to elevated temperatures before methylation results in racemization of aspartate and/or asparagine residues, and may result in isoaspartate formation as well. The methylatability of these samples also increases as a function of time of heating, independent of the pH (over the range pH 5-9) or Ca2+ concentration; the most significant increase occurs during the initial 60 min, when calmodulin retains a fraction of its biological activity. These results are consistent with the hypothesis that methylation of calmodulin may occur at these uncommon aspartate residues, but are not consistent with a regulatory role for the methylation reaction.

Age-related reductions in cerebral vasomotor reactivity and the law of initial value: a 4-year prospective longitudinal study.

A group of 51 neurologically normal, middle-aged and elderly volunteers (aged 35-86 years; mean age 63.24 years) with and without risk factors for stroke were given annual tests of cerebral vasomotor reactivity to assess any changes in the cerebral vascular capacitance associated with advancing age that might alter cerebral vasomotor reactivity. Cerebral vasomotor reactivity was estimated as the difference in bihemisphere gray matter CBF measured by the 133Xe inhalation method in the steady state breathing room air, followed by a second measurement during inhalation of 100% oxygen. There were significant and progressive reductions in cerebral vasomotor reactivity during the 4-year longitudinal study. Positive linear correlations were apparent between initial steady-state mean bihemisphere gray matter CBF levels and degrees of vasomotor reactivity, suggesting that the Law of Initial Value plays an important role. This should be borne in mind when analyzing scores of cerebral vasomotor reactivity. In the present communication, analysis of covariance was used to correct for influences of initial CBF levels on vasomotor responses tested while breathing pure oxygen.

Why are two different types of pyridine nucleotide transhydrogenase found in living organisms?

Two types of pyridine nucleotide transhydrogenases have been reported in living organisms. The energy-linked transhydrogenase is found in mitochondria and in certain heterotrophic and photosynthesizing bacteria, while the non-energy-linked transhydrogenase is found in certain heterotrophic bacteria. The presence of a structurally similar non-energy-linked transhydrogenase in Azotobacter vinelandii, Pseudomonas aeruginosa and Pseudomonas fluorescens is readily shown in extracts from these bacteria with Western (protein) blotting. This non-energy-linked enzyme is lacking in Escherichia coli, while the presence of a structurally similar energy-linked enzyme in E. coli and in beef heart mitochondria is indicated with the Western blotting technique. Spinach (Spinacia oleracea) lacks the non-energy-linked transhydrogenase occurring in bacteria. The chloroplast enzyme ferredoxin:NADP+ oxidoreductase, which exhibits non-energy-linked transhydrogenase activity, is immunologically distinct from the bacterial transhydrogenases. In order to provide a rationale for the distribution of the two types of pyridine nucleotide transhydrogenases, the steady-state degrees of reduction of the NADP(H) and NAD(H) pools in A. vinelandii (R'NADP(H) and R'NAD(H)) have been measured for cells metabolizing sucrose at a variable oxygen flux (phi O2). It is found that the degree of reduction of the NADP(H) pool is always higher than that of the NAD(H) pool (R'NADP(H) greater than R'NAD(H)) except when phi O2 goes to zero (R'NADP(H) approximately equal to R'NAD(H)). Comparison of these results with literature values indicates that the inequality R'NADP(H) greater than R'NAD(H) is always found in a membrane-enclosed compartment, irrespective of the type of transhydrogenase present. This allows an understanding of the function of the two types of pyridine nucleotide transhydrogenases in vivo. The physiological role of non-energy-linked transhydrogenase is to catalyze the reaction NADPH + NAD+ leads to NADP+ + NADH, that of energy-linked transhydrogenase to catalyze the reaction NADH + NADP+ leads to NADPH + NAD+. Since at equilibrium R'NADP(H) approximately equal to R'NAD(H) the inequality R'NADP(H) greater than R'NAD(H) under steady-state conditions explains the energy requirement in the latter reaction. The dependence of the non-energy-linked transhydrogenase activity of ferredoxin:NADP+ oxidoreductase on R'NADP(H) is compared with that of A, vinelandii transhydrogenase. The results indicate that this activity is unlikely to be of physiological importance in plant chloroplasts.

H.V.E.M. study of ordering and disordering in Cu3Au

Abstract Cu3Au has been irradiated with 600 kV electrons, and the rate of ordering and disordering determined between 140 and 400 K. The degree of order has been measured from the ratio of the intensities of the superlattice and fundamental reflections in electron diffraction patterns, these ratios having been calibrated under appropriate conditions using specimens of a known degree of order. At all irradiation temperatures a steady-state degree of order is developed. The results have been interpreted in terms of a model due to Zee and Wilkes (1980) with minor modifications. It has been found on this basis that the energy of migration of the defect responsible for ordering during irradiation is ∼0–62 eV, and this same value can be used to match the limited earlier data from neutron-damage studies.

Bistability in multiphoton ionization with recombination

The steady-state degree of ionization produced by multiphoton processes under bistable conditions is investigated. We show that the presence of recombination leads to a bistable steady state, with the degree of ionization abruptly increasing as the incident laser intensity I(i) is increased beyond a certain critical value I(i1) and abruptly decreasing as the intensity is decreased below a second critical value I(i2)<I(i1). Effects of dispersion on the bistability characteristics of the steady state are considered.

Pathogenesis of renal hyperchloremic acidosis resulting from dietary potassium restriction in the dog: role of aldosterone.

In dogs dietary K+ restriction (16 days) results in diminished urinary net acid excretion (NAE) and systemic hyperchloremic metabolic acidosis (sigma delta NAE, -200 meq; delta[HCO3-]p, -2.9 +/- 0.3 meq/liter, P less than 0.05). Urinary aldosterone (aldo) excretion decreased by 34 +/- 3% (P less than 0.001) and metabolic clearance rate of aldo increased by 80 +/- 17% (P less than 0.02) during K+ restriction. Daily subcutaneous injection of a small amount of exogenous aldo (20 micrograms) during K+ restriction significantly attenuated the reduction in NAE (sigma delta NAE -51 vs. -200 meq, P less than 0.05) without raising plasma aldo concentrations to levels greater than control. These findings suggest that hypoaldosteronism induced by potassium depletion is at least in part the cause of the observed renal tubular acidosis. In adrenalectomized (ADX) dogs maintained on fixed mineralocorticoid and glucocorticoid replacement (aldo dose 60 micrograms/day), K+ restriction resulted in a significant degree of renal metabolic acidosis (delta[HCO3-]p, -1.4 +/- 0.3 meq/liter, P less than 0.01). In these ADX dogs, the exogenous supply of aldo was fixed but hypoaldosteronism may have developed owing to increased metabolic clearance rate of aldo caused by dietary K+ depletion. When mineralocorticoid replacement was withheld in ADX dogs, the steady-state degree of renal metabolic acidosis was no more severe in animals with preexisting dietary K+ depletion (16 days) than in the same animals when mineralocorticoid was withheld without preexisting K+ depletion. Thus, when neither endogenous nor exogenous aldo is present, K+ depletion does not result in a renal acidosis-producing effect that exacerbates that of aldo deficiency. The results of these studies suggest that the reduction in NAE and consequent metabolic acidosis induced by dietary K+ depletion is at least in part a consequence of aldo deficiency, and provide no evidence of an additional defect in acidification not caused by aldo deficiency.

Intracellular oxygen utilization in mechanically overloaded rat heart.

Bilateral hypertrophy of the heart was induced in rats by aorto-caval fistula. One month after surgery, when the heart weight increased by about 60%, the hearts were excised and perfused according to Langendorff with bicarbonate buffer containing 2.0 mM pyruvate (pH 7.4, 37 °C, 75 Torr). Steady-state reduction degree of respiratory components as well as the state of myoglobin O2-saturation were monitored throughout the experiment in the visible range by means of a rapid scanning spectrophotometer. Double wavelength recordings of cytochrome aa3 and myoglobin absorption changes were done during a short period of anoxia (N2-equilibrated solution) followed by reoxygenation of the preparation (O2-equilibrated medium). The time course of cytochrome aa3 reoxidation was analysed with respect to intracellular pO2 changes as reflected by myoglobin saturation. The results of the above experiments suggest that cytochrome aa3 of overloaded hearts can be kept fully oxidized at lower intracellular pO2 (70 percent of Mb oxygenation) than in control hearts (90 percent of Mb oxygenation). A prospective mechanism of the observed phenomena as well as its impact on energy production of mechanically overloaded myocardium are briefly discussed.

Equilibrium chemical composition of the gases in a sealed high-pressure pulsed CO2laser

The dynamics of changes in the chemical composition of the gas mixture in a pulsed high-pressure gasdischarge CO2 laser with photopreionization, a high current-pulse repetition rate, and transverse circulation of the gas around a closed loop was studied and the setting up of an equilibrium chemical composition was investigated. The use of a high repetition frequency for the current pulses facilitated the rapid establishment of an equilibrium chemical composition in the gas mixture (in about 10–15 min), in a relatively large volume (60 liters), without the mixture being strongly contaminated by desorbed gases. It was established that after this interval of time, following the initiation of a discharge in the gas, a dynamic equilibrium was set up between the dissociation of the CO2 molecules and the recombination of the dissociation products. It was shown that a steady-state degree of dissociation of the CO2 gas was a function of the electric-discharge parameters and of the initial composition of the gas mixture, and was independent of the pressure. Under typical laser discharge conditions, the steady-state degree of dissociation of the CO2 molecules was 20–30%. It was found that hydrogen could be used as a catalyst for oxidizing carbon monoxide. On the basis of these investigations, the use of reduced pressures of the gas mixture is recommended for practically important operating regimes of pulsed CO2 lasers which employ gas mixtures in chemical equilibrium.

Original Article

Acetylsalicylic acid in daily doses from 3 to 6g was prescribed to 8 patients with rheumatoid arthritis. Various assessments of clinical effect, as well as steady-state plasma concentrations and degree of plasma protein binding, were determined at each dose. The unbound fraction of salicylate increased with the dose, resulting in very high free concentrations of drug in some patients. No statistically significant relationship between total or unbound plasma concentration and the measurements of clinical efficacy were obtained. The marked increase with the dose in unbound salicylate concentration is interpreted as the result of the saturable elimination known to occur for this drug.

ChemInform Abstract: METAL PHTHALOCYANINES USED AS CATALYSTS IN GAS PHASE REACTIONS. IX. OXIDATION N AND DECOMPOSITION OF 2‐PROPANETHIOL VAPOR CATALYZED BY CRYSTALLINE MONOMERIC β‐METAL PHTHALOCYANINES OF THE FIRST TRANSITION PERIOD

Abstract Monomeric β-metal phthalocyanines (MPc's) have been shown to be good catalysts for decomposition and oxidation of 2-propanthiol vapor at 250–350 °C. From the heats of activation and the degrees of conversion the following sequences of catalytic activity have been derived: FePc > CuPc > MnPc > CoPc > ZnPc > NiPc for the oxidation and CoPc > CuPc > FePc > ZnPc > MnPc, NiPc for the decomposition. Low heats of activation correspond to high degrees of conversion (FePc, CoPc) and vice versa (NiPc). Metal-free phthalocyanine is found to be inactive. H2S, formed by decomposition of 2-propanthiol, is strongly adsorbed; therefore, the steady-state degree of conversion of decomposition of 2-propanthiol is low. However, in the presence of O2, adsorbed H2S is oxidized readily forming SO2; the original catalytic activity of MPc's for decomposition of 2-propanthiol is thereby restored. For the same reason, the catalytic activity in oxidation of 2-propanthiol is influenced only to a moderate extent by H2S inhibition. Heats of activation are independent of the amount of H2S adsorbed; i.e., H2S reduces only the number of active centers involved in the reaction.

Metal phthalocyanines used as catalysts in gas phase reactions: IX. Oxidation and decomposition of 2-propanthiol vapor catalyzed by crystalline monomeric β-metal phthalocyanines of the first transition period

Monomeric β-metal phthalocyanines (MPc's) have been shown to be good catalysts for decomposition and oxidation of 2-propanthiol vapor at 250–350 °C. From the heats of activation and the degrees of conversion the following sequences of catalytic activity have been derived: FePc > CuPc > MnPc > CoPc > ZnPc > NiPc for the oxidation and CoPc > CuPc > FePc > ZnPc > MnPc, NiPc for the decomposition. Low heats of activation correspond to high degrees of conversion (FePc, CoPc) and vice versa (NiPc). Metal-free phthalocyanine is found to be inactive. H 2S, formed by decomposition of 2-propanthiol, is strongly adsorbed; therefore, the steady-state degree of conversion of decomposition of 2-propanthiol is low. However, in the presence of O 2, adsorbed H 2S is oxidized readily forming SO 2; the original catalytic activity of MPc's for decomposition of 2-propanthiol is thereby restored. For the same reason, the catalytic activity in oxidation of 2-propanthiol is influenced only to a moderate extent by H 2S inhibition. Heats of activation are independent of the amount of H 2S adsorbed; i.e., H 2S reduces only the number of active centers involved in the reaction.

Pharmacokinetics and pharmacodynamics of alprenolol in the treatment of hypertension. II. Relationship to its effect on blood pressure and plasma renin activity.

The kinetics of alprenolol, in relation to its effect on blood pressure and plasma renin activity, have been studied in sixteen patients. A within-patient comparison was made between therapy for six weeks with placebo or alpronolol 200 mg thrice daily. Thirteen patients responded to alprenolol by a signoficant fall in blood pressure. In three other patients treatment did not lower blood pressure. In the group as a whole there was no significant correlation between the fall in systolic, diastolic or mean blood pressure, and the steady-state plasma alprenolol concentration, renin status, or degree of beta blockade. However, the thirteen responsive patients showed a significant relationship (p less than 0.05 - 0.001) between the log mean steady-state plasma alprenolol concentration and the hypotensive response.

Oxidation of cytochrome b5 by hydroperoxides in rat liver.

1. Spectral changes following the addition of hydroperoxides to isolated hepatocytes and to perfused rat liver were observed. Cytochrome b5 is the major, if not the only, hemoprotein exhibiting redox changes under these conditions; cytochrome b5 is oxidized by added hydroperoxides, e.g. tert-butyl or cumene hydroperoxides. No spectral changes attributable to cytochrome b5 were observed with tert-butanol. 2. The effect is present also when the mitochondrial respiratory chain is inhibited by antimycin A, and it is not observable with isolated mitochondria. On the other hand, the oxidation of cytochrome b5 by hydroperoxides is readily demonstrable in microsomal fractions in presence of NADH. 3. Spectral evidence for a participation of the other microsomal hemoprotein, cytochrome P-450, in the hydroperoxide-linked effects was not obtained. Thus, in hepatocytes from phenobarbital-pretreated rats, no formation of cytochrome P-420, no displacement of a type I substrate, hexobarbital, and no major steady state redox change of cytochrome P-450 was detectable. However, when cytochrome P-450 was dithionite-reduced, an oxidation of this cytochrome occurred upon subsequent hydroperoxide addition. 4. Hydrogen peroxide addition to hepatocytes also leads to a lower steady-state degree of reduction of cytochrome b5. Evidence is provided with hepatocytes from rats pretreated with 3-amino-1,2,4-triazole that H2O2 generated intracellularly, e.g. from added glycolate, also causes a detectable oxidation of cytochrome b5. 5. The mechanism of these hydroperoxide effects remains to be established, and it is not clear whether cytochrome b5 reacts directly or indirectly. However, it is suggested that these effects may be of significance for the further study of cytochrome-b5-linked metabolic pathways.

The influence of sulphur compounds on β-Cu-phthalocyanine used as an oxidation catalyst

Monomeric β-metal phthalocyanines (MPc's) have been shown to be good catalysts for decomposition and oxidation of 2-propanthiol vapor at 250–350 °C. From the heats of activation and the degrees of conversion the following sequences of catalytic activity have been derived: FePc > CuPc > MnPc > CoPc > ZnPc > NiPc for the oxidation and CoPc > CuPc > FePc > ZnPc > MnPc, NiPc for the decomposition. Low heats of activation correspond to high degrees of conversion (FePc, CoPc) and vice versa (NiPc). Metal-free phthalocyanine is found to be inactive. H2S, formed by decomposition of 2-propanthiol, is strongly adsorbed; therefore, the steady-state degree of conversion of decomposition of 2-propanthiol is low. However, in the presence of O2, adsorbed H2S is oxidized readily forming SO2; the original catalytic activity of MPc's for decomposition of 2-propanthiol is thereby restored. For the same reason, the catalytic activity in oxidation of 2-propanthiol is influenced only to a moderate extent by H2S inhibition. Heats of activation are independent of the amount of H2S adsorbed; i.e., H2S reduces only the number of active centers involved in the reaction.

Outward membrane currents activated in the plateau range of potentials in cardiac Purkinje fibres.

1. The membrane currents in Purkinje fibres under voltage clamp conditions have been investigated in the range of potentials at which the action potential plateau occurs. The results show that in this range slow outward current changes occur which are quite distinct from the potassium current activated in the pace-maker range of potentials.2. The time course of current change in response to step voltage changes is non-exponential. At each potential the current changes may be analysed in terms of the sum of two exponential changes and this property has been used to dissect the currents into two components, i(x1) and i(x2), both of which have been found to obey kinetics of the Hodgkin-Huxley type.3. The first component, i(x1), is activated with a time constant of about 0.5 sec at the plateau. At more positive and more negative potentials the time constants are shorter. The steady-state degree of activation varies from 0 at about -50 mV to about 1 at +20 mV. The instantaneous current-voltage relation is an inward-going rectifier but shows no detectable negative slope. In normal Tyrode solution ([K](0) = 4 mM) the reversal potential is about -85 mV.4. The second component, i(x2), is activated extremely slowly and the time constant at the plateau is about 4 sec. The steady-state activation curve varies from 0 at about -40 mV to 1 at about +20 mV. The instantaneous current-voltage relation is nearly linear. The reversal potential occurs between -50 and -20 mV in different preparations.5. It is suggested that these currents are carried largely by K ions, but that some other ions (e.g. Na) also contribute so that the reversal potentials are positive to E(K).6. The relation of these results to previous work on delayed rectification in cardiac muscle is discussed.