scholarly journals Inhibition of fly head acetylcholinesterase by bis-[(m-hydroxyphenyl)trimethylammonium iodide] esters of polymethylenedicarbamic acids

1970 ◽  
Vol 117 (2) ◽  
pp. 221-230 ◽  
Author(s):  
J. H. Davies ◽  
W. R. Campbell ◽  
C. W. Kearns
Keyword(s):  
Functional Group ◽  
Straight Chain ◽  
Experimental Conditions ◽  
Maximum Slope ◽  
Large Molecules ◽  
High Affinity ◽  
Affinity Constants ◽  
Low Concentrations ◽  
High Concentrations ◽  
Pass Through

A series of bis-[(m-hydroxyphenyl)trimethylammonium iodide] esters of polymethylenedicarbamic acids and a number of (m-hydroxyphenyl)trimethylammonium iodide esters of straight-chain N-alkylcarbamic acids have been examined as inhibitors of acetylcholinesterase from fly head. Evidence is presented suggesting that inhibition of acetylcholinesterase by the bis-carbamates is due to carbamoylation of the enzyme, as is generally thought to be the case with esters of N-alkylcarbamic acids. Inhibition is irreversible. The (m-hydroxyphenyl)trimethylammonium iodide ester of N-hexylcarbamic acid also inhibits fly head acetylcholinesterase irreversibly. There is therefore no need to implicate a second functional group in bis-carbamate esters to explain the irreversible inhibition of the enzyme. An unusual feature of the inhibition is that inhibition lines do not pass through 100% enzyme activity at t=0, except for rather low concentrations of inhibitor (<10μm for the octamethylene compound). Also, inhibition lines tend towards a maximum slope as inhibitor concentration is increased. The first observation indicates complex-formation, even in the presence of high concentrations of substrate, and by using measurements of inhibition at relatively high inhibitor concentrations, affinity constants K′a have been calculated. K′a varies from 0.1μm for the dodecamethylene compound to 10μm for the tetramethylene compound, in the presence of 3.75mm-acetylthiocholine, indicating high affinity for the enzyme. The second observation shows that, owing to this high affinity, the enzyme becomes saturated with inhibitor under the experimental conditions employed, and from the limiting slope values of the carbamoylation rate constant (k2) have been calculated. k2 varies from 0.15min−1 for the tetramethylene compound to 1min−1 for the decamethylene compound. Variations of potency in this series are therefore mainly due to changes in affinity (100-fold) rather than in carbamoylation rate (sevenfold). The observation that large molecules may acylate the enzyme raises certain problems, which are discussed.

scholarly journals The Relation of the Composition of the Culture Medium to the Formation of Endospores by Aerobic Bacilli

1932 ◽  
Vol 32 (4) ◽  
pp. 535-543 ◽  
Author(s):  
H. L. A. Tarr
Keyword(s):  
Amino Acids ◽  
Culture Medium ◽  
Inorganic Salt ◽  
Synthetic Medium ◽  
Ammonium Phosphate ◽  
Spore Formation ◽  
Experimental Conditions ◽  
Nitrogenous Compounds ◽  
Low Concentrations ◽  
High Concentrations

1. Spore formation in eight typical members of the genusBacillushas been studied.2. Three of these strains, including one species ofB. anthracis, have been found to be practically asporogenous under the experimental conditions. In general the following statements hold good for the sporogenous races studied.3. Spore formation is almost, or entirely, inhibited by cultivation on media rich in amino acids, such as tryptic digests of casein or meat. Similar inhibition results following cultivation on a medium containing reasonably high concentrations of a mixture of amino acids and asparagine.4. When such media are suitably diluted with standard inorganic salt solutions the percentage of spores formed is greatly increased, and frequently at least 99 per cent. of spores are formed if the dilution is sufficiently high.5. When simple nitrogenous compounds such as amino acids are added to a dilute casein digest medium in which sporulation is almost complete, a definite decrease in the percentage of spores present is observed. Asparagine, which is probably readily assimilated, apparently completely hinders spore formation in most cases. Other amino acids do not exert so pronounced an effect, and ammonium phosphate does not appreciably inhibit the formation of spores.6. The fact that the addition of glycine suppresses growth markedly when it is added to a dilute casein digest medium, but does not appreciably hinder sporulation, suggests that the formation of spores is not due to any toxic effect of added compounds, or compounds already present in the medium.7. Sporulation is almost complete in a “synthetic” medium in which low concentrations of ammonium phosphate and sucrose represent the sources of nitrogen and carbon, respectively. However, frequent transfers in such a medium may inhibit spore formation partially or entirely in certain instances. This effect probably depends upon the enhanced ability of the culture in question to utilise sucrose as a source of carbon when cultivated constantly in its presence.8. It is concluded, from the above data, that endospore formation in aerobic bacilli bears an inverse relationship to the amount of available nutrient material present in the culture medium.I am indebted to Prof. Sir F. G. Hopkins and Miss M. Stephenson for their constant encouragement during the progress of this work. My thanks are due to Mr Pirie of this Department who kindly furnished me with several of the amino acids employed, and to Dr Miles of the Department of Pathology for his kindness in supplying me with certain of the cultures.

scholarly journals Monoclonal antibody defining a molecule possibly identical to the p75 subunit of interleukin 2 receptor.

1989 ◽  
Vol 169 (4) ◽  
pp. 1323-1332 ◽  
Author(s):  
T Takeshita ◽  
Y Goto ◽  
K Tada ◽  
K Nagata ◽  
H Asao ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Interleukin 2 ◽  
High Affinity ◽  
Human T Cell ◽  
Low Concentrations ◽  
High Concentrations ◽  
Dependent Growth ◽  
T Cell Lines

A mouse hybridoma cell line, TU27, producing an mAb was established. TU27 mAb reacted with various human and Gibbon ape T cell lines bearing the IL-2R p75 (IL-2Rp75), but not with cell lines expressing only Tac antigen, IL-2Rp55, and numbers of its binding sites on cell surfaces were similar to those of high-affinity IL-2R. Radioimmunoprecipitation with TU27 mAb defined a molecule with a molecular mass of 75 kD on the surface of IL-2Rp75 bearing cells. TU27 mAb completely blocked IL-2 binding to IL-2Rp75 and to the high-affinity IL-2R but not to IL-2Rp55 composing the low-affinity IL-2R. The IL-2-dependent growth of a human T cell line, ILT-Mat, was significantly inhibited by TU27 mAb only at low concentrations of IL-2, and combination of TU27 mAb and H-31 mAb specific for IL-2Rp55 completely inhibited the cell growth even at high concentrations of IL-2. These data strongly suggest that TU27 mAb is specific for the human IL-2Rp75.

scholarly journals Steady-state kinetics of catecholamine transport by chromaffin-granule ‘ghosts’

1974 ◽  
Vol 144 (2) ◽  
pp. 319-325 ◽  
Author(s):  
J H Phillips
Keyword(s):  
Steady State ◽  
Competitive Inhibitor ◽  
Affinity Constant ◽  
Chromaffin Granule ◽  
Affinity Constants ◽  
Steady State Kinetics ◽  
Low Concentrations ◽  
High Concentrations ◽  
Chromaffin Granule Ghosts ◽  
Kinetics Of

Resealed chromaffin-granule ‘ghosts’ were used to study the steady-state kinetics of catecholamine transport. The pump has a high affinity for (-)-noradrenaline, (-)-adrenaline, tyramine and 5-hydroxytryptamine (serotonin), but a lower affinity for (+)-noradrenaline. The measured rates of incorporation do not conform to Michaelis–Menten kinetics, but affinity constants for the former substrates are in the range 8–18μm. Reserpine is a potent inhibitor. Incorporation as a function of ATP concentration also fails to show simple kinetics; the affinity constant for ATP is deduced to be about 3mm at 1mm-MgCl2. Adenylyl (βγ-methylene)diphosphonate is a competitive inhibitor at low concentrations, but inhibits more strongly at high concentrations. The pump has a transition temperature at 29°C and does not seem to be identical with the Mg2+-stimulated adenosine triphosphatase of chromaffin granules.

scholarly journals Differentiation of Amphetamine/Methamphetamine and Other Cross-Immunoreactive Sympathomimetic Amines in Urine Samples by Serial Dilution Testing

2006 ◽  
Vol 52 (4) ◽  
pp. 743-746 ◽  
Author(s):  
Alison Woodworth ◽  
Al N Saunders ◽  
John W Koenig ◽  
Thomas P Moyer ◽  
John Turk ◽  
...  
Keyword(s):  
Dose Response ◽  
Roc Analysis ◽  
Urine Samples ◽  
Gas Chromatography Mass Spectrometry ◽  
Maximum Slope ◽  
Patient Disposition ◽  
Low Concentrations ◽  
High Concentrations ◽  
Dynamic Assay ◽  
Urine Specimens

Abstract Background: Immunoassay-based screening for amphetamines has a variable positive predictive value (PPV) for detecting amphetamine abuse. The lack of immunoassay specificity necessitates confirmatory testing by gas chromatography–mass spectrometry (GC/MS), but the technical complexity and expense of GC/MS limit its availability. Physicians may make decisions regarding patient disposition based on unverified results. In this study we assessed the utility of using dose–response properties to distinguish urine samples containing amphetamines from samples containing cross-immunoreactive species. Methods: Urine was supplemented with known concentrations of amphetamine, methamphetamine, methylenedioxymethamphetamine (MDMA), or pseudoephedrine. Using a series of dilutions, we determined the maximum change in rate over the fractional change in concentration for each compound in the Emit® II amphetamine/methamphetamine immunoassay. Patient urine samples that screened positive for amphetamines were diluted 1:1, 1:10, and 1:20, and maximum slope estimates within the dynamic assay range were determined. An optimal slope cutoff that differentiated samples containing (meth)amphetamine from those containing cross-reacting species was determined by ROC analysis. Results: The slope of the dose response was largest for amphetamine and methamphetamine, followed by MDMA and pseudoephedrine. The optimum slope cutoff for identifying patient specimens containing (meth)amphetamine was 320 (sensitivity, 96%; specificity, 90%; PPV, 92%). High concentrations of less reactive compounds may mask low concentrations of amphetamines. Conclusions: Use of the slope of the dose–response relationship in patient urine specimens can enhance the PPV of presumptive positive immunoassay results but does not exclude the presence of low amphetamine concentrations in samples containing high concentrations of cross-reactive species.

Characterization of the high and low affinity components of the renal Ca2+ – Mg2+ ATPase

1990 ◽  
Vol 68 (6) ◽  
pp. 718-726 ◽  
Author(s):  
M. G. Brunette ◽  
J. Mailloux ◽  
M. Chan ◽  
C. Ramachandran
Keyword(s):  
Enzyme Activity ◽  
Atp Hydrolysis ◽  
Free Calcium ◽  
Atp Binding ◽  
High Affinity ◽  
Atp Concentration ◽  
Low Concentrations ◽  
High Affinity Site ◽  
High Concentrations

The purpose of this study was to characterize the interrelationship between free calcium (Ca2+) and magnesium (Mg2+) in the Ca2+ ATPase enzyme cycle of kidney membranes. Experiments were performed with basolateral membranes from rat renal cortex and microdissected proximal and distal tubules from mice. Results were similar in the three types of preparations. We first investigated the effect of ATP concentration on Ca2+- and Mg2+-dependent ATP hydrolysis. With 0.2 μM Ca2+, the enzyme activity, as a function of ATP concentration, showed two saturable components: a high affinity component with a Km of 33 μM ATP and a low affinity component with a Km of 0.63 mM ATP. These components may represent either two distinct sites of ATP binding or two forms of the same site. For the sake of simplicity, it was assumed that the two components correspond to a high affinity and a low affinity substrate site. At the high affinity site (ATP = 50 μM), the Ca2+ dependence of ATP hydrolysis followed a single Michaelis–Menten kinetics with Km for Ca2+ of 0.08 μM. The addition of 1 mM Mg2+ resulted in a relatively constant increase in ATP hydrolysis at all Ca2+ concentrations, indicating that the effects of the two cations were additive. With high ATP concentration (ATP = 3 mM), Ca2+ also induced an ATP hydrolysis according to a saturable process, with a Km for Ca2+ of 0.2 μM. In contrast with what occurred with low concentrations of ATP, addition of millimolar Mg2+ completely curtailed the sensitivity of the enzyme to Ca2+. However, traces of Mg2+ (< 10−6 M), while decreasing the Vmax of the Ca2+ ATPase, increased the affinity of the enzyme for Ca2+. These results indicate that with high concentrations of ATP, Mg2+ not only modifies the enzyme conformation but also displaces Ca2+. The effects of calmodulin, vanadate, and lanthanum were also examined using low and high concentrations of ATP. With 50 μM ATP, neither 0.5 μM calmodulin nor 100 μM lanthanum had any influence on the Ca2+-dependent ATP hydrolysis; in contrast, 100 μM vanadate significantly diminished the enzyme activity. With 3 mM ATP, 0.5 μM calmodulin increased the affinity of the enzyme for Ca2+, whereas 100 μM vanadate decreased it. These results suggest that the effect of calmodulin occurs only when the two sites are occupied, whereas vanadate interferes in presence of both low and high ATP concentrations. Based on the analogy with the sequential elementary steps described for other membrane Ca2+ ATPases and for Na+–K+ ATPase, and on the similarities between these characteristics of ATP hydrolysis and those of the enzyme phosphorylation on one hand and ATP-dependent Ca2+ transport on the other hand, we propose that the phosphoenzyme formation occurs at the high affinity site and that Ca2+ transport is directly related to ATP binding at the low affinity site, which might be considered as a regulatory site.Key words: kidney Ca2+ ATPase, ATP magnesium and calcium on Ca2+ ATPase, ATP-dependent calcium transport in kidney.

The Modification of Actomyosin ATPase Activity by Tropomyosin-Troponin and its Dependence on Ionic Strength, ATP-Concentration, and Actin-Myosin Ratio

1974 ◽  
Vol 29 (9-10) ◽  
pp. 496-505 ◽  
Author(s):  
Peter Daneker
Keyword(s):  
Ionic Strength ◽  
Atpase Activity ◽  
Actin Filaments ◽  
Regulatory Proteins ◽  
High Affinity ◽  
Low Concentrations ◽  
Dependence On Ionic Strength ◽  
High Concentrations ◽  
Low Ionic Strength ◽  
Better Than

Abstract At millimolar concentrations of ATP the ATPase activity of regulated actomyosin (which consisted of myosin and of actin containing the regulatory proteins tropomyosin and troponin) was lower than that of unregulated actomyosin (containing actin devoid of the regulatory proteins) when the ionic strength was high (> 0 .0 3 ᴍ KCl). At low ionic strength (0.03 ᴍ KCl) the ATPase activity of regulated actomyosin was similar to or even higher than that of unregulated acto­ myosin. Besides increasing ionic strength an increasing actin-myosin ratio tended to depress the ATPase activity of regulated actomyosin below that of unregulated one. At lower ATP concen­ trations (0.1 mᴍ or lower) the ATPase activity of regulated actomyosin was higher than that of unregulated actomyosin at any ionic strength and at any actin-myosin ratio. EGTA inhibited the ATPase of regulated actomyosin under any conditions at high ATP concentrations. At lower ATP concentrations EGTA inhibited either at higher ionic strength or at a higher actin-myosin ratio. The inhibition of the ATPase activity of acto-HMM by increasing ionic strength was not in­ fluenced by the regulatory proteins. - For the interpretation of these results it has been assumed that in actomyosin regulated actin can adopt three states: A low-affinity state which activates the ATPase of myosin only slightly (occurring at high ATP concentrations and in the absence of Ca2+), a high affinity state which activates the ATPase of myosin better than does unregulated actin (occurring at low concentrations of ATP and in the presence of Ca2+), and an intermediate state. This latter state (occurring at high concentrations of ATP and in the presence of Ca2+ or at low concentrations of ATP and in the absence of Ca2+) activates the ATPase of myosin less than does unregulated actin when the actin-myosin ratio is high (wide spacing of myosin on the actin filaments) but activates more (or at least not less) when the actin-myosin ratio is low (dense spacing of myosin on the actin filaments)

Neutrophil Elastase Potentiates Cathepsin G-lnduced Platelet Activation

1992 ◽  
Vol 68 (05) ◽  
pp. 570-576 ◽  
Author(s):  
Mary A Selak
Keyword(s):  
Neutrophil Elastase ◽  
Cell Activation ◽  
Thromboxane A2 ◽  
Creatine Phosphate ◽  
Dense Granule ◽  
Cathepsin G ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Low Concentrations ◽  
High Concentrations

SummaryWe have previously demonstrated that human neutrophil cathepsin G is a strong platelet agonist that binds to a specific receptor. This work describes the effect of neutrophil elastase on cathepsin G-induced platelet responses. While platelets were not activated by high concentrations of neutrophil elastase by itself, elastase enhanced aggregation, secretion and calcium mobilization induced by low concentrations of cathepsin G. Platelet aggregation and secretion were potentiated in a concentration-dependent manner by neutrophil elastase with maximal responses observable at 200 nM. Enhancement was observed when elastase was preincubated with platelets for time intervals of 10–60 s prior to addition of a low concentration of cathepsin G and required catalytically-active elastase since phenylmethanesulphonyl fluoride-inhibited enzyme failed to potentiate cell activation. Neutrophil elastase potentiation of platelet responses induced by low concentrations of cathepsin G was markedly inhibited by creatine phosphate/creatine phosphokinase and/or indomethacin, indicating that the synergism between elastase and cathepsin G required the participation of ADP and thromboxane A2. On the other hand, platelet responses were not attenuated by the PAF antagonist BN 52021, signifying that PAF-acether did not play a role in elastase potentiation. At higher concentrations porcine pancreatic elastase exhibits similar effects to neutrophil elastase, demonstrating that the effect of elastase was not unique to the neutrophil protease. While neutrophil elastase failed to alter the ability of cathepsin G to hydrolyze a synthetic chromogenic substrate, preincubation of platelets with elastase increased the apparent affinity of cathepsin G binding to platelets. In contrast to their effect on cathepsin G-induced platelet responses, neither neutrophil nor pancreatic elasatse potentiated aggregation or dense granule release initiated by ADP, PAF-acether, arachidonic acid or U46619, a thromboxane A2 mimetic. Moreover, unlike its effect on cathepsin G, neutrophil elastase inhibited thrombin-induced responses. The current observations demonstrate that elastase can potentiate platelet responses mediated by low concentrations of cathepsin G, suggesting that both enzymes may function synergistically to activate platelets under conditions where neutrophil degranulation occurs.

In Vitro Effects of Ethanol on Rabbit Platelet Aggregation, Secretion of Granule Contents, and Cyclic AMP Levels in the Presence of Prostacyclin

1989 ◽  
Vol 61 (02) ◽  
pp. 254-258 ◽  
Author(s):  
Margaret L Rand ◽  
Peter L Gross ◽  
Donna M Jakowec ◽  
Marian A Packham ◽  
J Fraser Mustard
Keyword(s):  
Cyclic Amp ◽  
Inhibitory Effect ◽  
Rabbit Platelet ◽  
Inhibitory Effects ◽  
Low Concentrations ◽  
Rabbit Platelets ◽  
High Concentrations ◽  
In Vitro Effects ◽  
Aggregation Of Platelets

SummaryEthanol, at physiologically tolerable concentrations, inhibits platelet responses to low concentrations of collagen or thrombin, but does not inhibit responses of washed rabbit platelets stimulated with high concentrations of ADP, collagen, or thrombin. However, when platelet responses to high concentrations of collagen or thrombin had been partially inhibited by prostacyclin (PGI2), ethanol had additional inhibitory effects on aggregation and secretion. These effects were also observed with aspirin- treated platelets stimulated with thrombin. Ethanol had no further inhibitory effect on aggregation of platelets stimulated with ADP, or the combination of ADP and epinephrine. Thus, the inhibitory effects of ethanol on platelet responses in the presence of PGI2 were very similar to its inhibitory effects in the absence of PGI2, when platelets were stimulated with lower concentrations of collagen or thrombin. Ethanol did not appear to exert its inhibitory effects by increasing cyclic AMP above basal levels and the additional inhibitory effects of ethanol in the presence of PGI2 did not appear to be brought about by further increases in platelet cyclic AMP levels.

Aggregation of Cat Platelets in Vitro

1970 ◽  
Vol 23 (03) ◽  
pp. 601-620 ◽  
Author(s):  
Th. B Tschopp
Keyword(s):  
Adenosine Monophosphate ◽  
Blood Collection ◽  
Base Line ◽  
Reversible Aggregation ◽  
Low Concentrations ◽  
Irreversible Aggregation ◽  
High Concentrations ◽  
Two Phases ◽  
Improved Technique

SummaryAggregation of cat platelets in the citrated plasma is examined by means of Born’s absorptiometer. A marked tendency of the platelets of this species to spontaneous aggregation necessitated first of all the development of an improved technique of blood collection.A hypothesis according to which 5-HT is released from the platelets, explains the absence of oscillations on the base line of the absorptiometer, the absence of platelet swelling, when ADP is added, and the effect of stirring on the aggregation curves in cat PRP. The average volume of cat platelets amounts to 10.46 μ3 when directly fixed in the blood, when fixed from PRP to 12.17 μ3, when fixed from stirred PRP to 13.51 μ3.In low concentrations (0.3-2 μM) ADP produce reversible aggregation; in narrowly restricted, individually dissimilar mean concentrations irreversible aggregation in two phases and in high concentrations, irreversible aggregation in one phase. Like ADP serotonin produces 2 phase irreversible aggregation in concentrations of 3-10 μM, but unlike ADP, the aggregation velocity decreases again with high 5-HT concentrations (>100 μM). Adrenaline does not produce aggregation and it is likely that adenosine and adenosine monophosphate inhibit the aggregation by serotonin but not by ADP. Species differences in the aggregation of human, rabbit and cat platelets are discussed.

The Action of an Aniline Derivative (AN 162) on Blood Coagulation and on Platelets

1971 ◽  
Vol 26 (01) ◽  
pp. 145-166
Author(s):  
E Deutsch ◽  
K Lechner ◽  
K Moser ◽  
L Stockinger
Keyword(s):  
Blood Coagulation ◽  
Citric Acid Cycle ◽  
Second Phase ◽  
Aniline Derivative ◽  
Acid Cycle ◽  
Enhancing Effect ◽  
Low Concentrations ◽  
Dual Action ◽  
High Concentrations ◽  
Induced Aggregation

Summary1. The aniline derivative AN 162, Donau Pharmazie, Linz, Austria, has a dual action on the blood coagulation: an anticoagulant and an coagulation enhancing effect.2. The anticoagulant action may only be demonstrated with high concentrations (over 1 X 10”3 M related to plasma) preferentially in PPP. It is partially caused by an inhibition of the endogenous way of generation of the prothrombin converting principle. In addition it is suggested that it interferes with the fibrinogen-fibrin reaction in a manner not yet understood.3. The coagulant action is caused by a greater availability of platelet constituents at low concentrations of AN 162 (over 1 × 10-4 M) and by the induction of a release reaction at higher concentrations. The platelet factors 3 and 4, serotonin, adenine, and acid phosphatase are released.4. AN 162 inhibits platelet aggregation. This inhibition can be demonstrated by the PAT of Breddin and in the stirred aggregation test of Born. It is more effective to inhibit the collagen-induced and the second phase of the adrenaline-induced aggregation than the ADP induced one. The platelet retention (test of Hellem) is also reduced.5. The action of AN 162 on the platelets is caused by a damage of the platelet membrane which becomes permeabel for both, soluble platelet constitutents and granula.6. AN 162 interferes with the energy metabolism of the platelets. It causes a loss of ATP, and inhibits the key-enzymes of glycolysis, citric acid cycle, fatty acid oxydation and glutathione reduction.7. AN 162 inhibits the growth of fibroblasts without influence on mitosis.

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