Isolated flagellar apparatus of Chlamydomonas: characterization of forward swimming and alteration of waveform and reversal of motion by calcium ions in vitro

1978 ◽  
Vol 33 (1) ◽  
pp. 235-253 ◽  
Author(s):  
J.S. Hyams ◽  
G.G. Borisy
Keyword(s):  
Calcium Ions ◽  
Beat Frequency ◽  
Flagellar Apparatus ◽  
Adenosine Monophosphate ◽  
Adenosine Diphosphate ◽  
Living Cells ◽  
Exogenous Calcium ◽  
Ph Range ◽  
Relationship Of

The control of flagellar activity in the biflagellate green alga, Chlamydomonas reinhardtii was investigated by the in vitro reactivation of the isolated flagellar apparatus (the 2 flagella attached to their respective basal bodies plus accessory structures). The waveform and beat frequency of the isolated apparatus in the presence of 1 mM adenosine triphophate (ATP) were comparable to those recorded for living cells. Equimolar concentrations of adenosine diphosphate (ADP) could be substituted for ATP with little change in beat frequency and no apparent change in waveform, suggesting that the latter is converted to ATP by axonemal adenylate kinase. No reactivation occurred in adenosine monophosphate (AMP). But frequencies in cytidine, guanosine and uridine triphosphates (CTP, GTP and UTP) were approximately 10% that obtained in ATP. Reactivation was optimal over a broad pH range between pH 6.4 and pH 8.9 in both APT and ADP. Isolated flagellar apparatus could be induced to change from forward to reverse motion in vitro by manipulation of exogenous calcium ions. The 2 types of motion were directly comparable to recorded responses of living cells. Forward swimming occurred at levels of calcium below 10(−6)M, the isolated apparatus changing to backward motion above this level. Motility was inhibited at concentrations above 10(−3)M. The threshold for reversal of motion by calcium was lowered to 10(−7)M when the flagellar membranes were solubilized with detergent, indicating that the flagellar membranes are involved in the regulaion of the level of calcium within the axoneme. The reversal of motion by calcium was itself freely reversible. The relationship of these observations to the known tactic responses of Chlamydomonas is discussed.

scholarly journals SIMILARITY OF THE KINETICS OF INVERTASE ACTION IN VIVO AND IN VITRO. II

1932 ◽  
Vol 16 (2) ◽  
pp. 233-242 ◽  
Author(s):  
B. G. Wilkes ◽  
Elizabeth T. Palmer
Keyword(s):  
Physiological Activity ◽  
Outer Region ◽  
Living Cells ◽  
Yeast Cells ◽  
Live Cells ◽  
Intracellular Enzyme ◽  
Relationship Of ◽  
Kinetics Of

1. The pH-activity relationship of invertase has been studied in vivo and in vitro under identical external environmental conditions. 2. The effect of changing (H+) upon the sucroclastic activity of living cells of S. cerevisiae and of invertase solutions obtained therefrom has been found, within experimental error, to be identical. 3. The region of living yeast cells in which invertase exerts its physiological activity changes its pH freely and to the same extent as that of the suspending medium. It is suggested that this may indicate that this intracellular enzyme may perform its work somewhere in the outer region of the cell. 4. In using live cells containing maltase, no evidence of increased sucroclastic activity around pH 6.9, due to the action of Weidenhagen's α-glucosidase (maltase), was found.

Inhibition of H+ secretion in the frog gastric mucosa by ATP and related compounds

1976 ◽  
Vol 230 (6) ◽  
pp. 1688-1694 ◽  
Author(s):  
SS Sanders ◽  
CF Butler ◽  
J O'Callaghan ◽  
WS Rehm
Keyword(s):  
Gastric Mucosa ◽  
Rana Pipiens ◽  
Adenosine Monophosphate ◽  
Adenosine Diphosphate ◽  
Threshold Concentration ◽  
Maximal Inhibition ◽  
Secretory Rate ◽  
Free Media ◽  
Related Compounds

Addition of adenosine triphosphate (ATP) to the nutrient (submucosal-facing) solution of the histamine-stimulated in vitro frog (Rana pipiens) gastric mucosa produces a marked reduction in the H+ secretory rate and an increase in transmucosal potential difference (PD) and resistance in both Cl- and Cl-free media. The effects are reversible upon removal of ATP. The threshold concentration is between 1 and 2 mM, and 5 mM produce maximal inhibition. It is shown that the effects of ATP are not due to a change in pH or osmolarity of the nutrient fluid, or to a decrease in the Ca2+ and/or Mg2+ activities of the nutrient fluid. It is also shown that the inhibitory action of ATP is not dependent on a chelation complex between Ca2+ or Mg2+. Adenosine diphosphate also produces effects essentially the same as ATP whereas 5'-adenosine monophosphate and adenosine produce relatively little or no change.

scholarly journals Comparative In Vitro Study of Various α2-Adrenoreceptor Agonist Drugs for Ticagrelor Reversal

2020 ◽  
Vol 9 (3) ◽  
pp. 809
Author(s):  
Guillaume Porta Bonete ◽  
Anne Godier ◽  
Pascale Gaussem ◽  
Tiphaine Belleville-Rolland ◽  
Alexandre Leuci ◽  
...  
Keyword(s):  
In Vitro Study ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Adenosine Diphosphate ◽  
Camp Pathway ◽  
P2y12 Receptor Antagonist ◽  
Induced Aggregation ◽  
Camp Levels ◽  
Platelet Functions

Ticagrelor, an antiplatelet adenosine diphosphate (ADP)-P2Y12 receptor antagonist, increases the risk of bleeding. Its management is challenging because platelet transfusion is ineffective and no specific antidote is currently available. Epinephrine, a vasopressor catecholamine prescribed during shock, restores platelet functions inhibited by ticagrelor through stimulation of α2A-adrenoreceptors. It subsequently inhibits cyclic adenosine monophosphate (cAMP) pathway and PI3K signaling. However, since epinephrine may expose a patient to deleterious hemodynamic effects, we hypothesized that other α2-adrenoreceptor agonist drugs used in clinical practice with fewer side effects could reverse the antiplatelet effects of ticagrelor. We compared in vitro the efficacy of clonidine, dexmedetomidine, brimonidine, and norepinephrine with epinephrine to restore ADP- and PAR-1-AP-induced washed platelet aggregation inhibited by ticagrelor, as well as resulting platelet cAMP levels. In ticagrelor-free samples, none of the α2-adrenoreceptor agonists induced aggregation by itself but all of them potentiated ADP-induced aggregation. Compared with epinephrine, norepinephrine, and brimonidine partially restored ADP- and fully restored PAR-1-AP-induced aggregation inhibited by ticagrelor while clonidine and dexmedetomidine were ineffective. Indeed, this lack of effect resulted from a lower decrease in cAMP concentration elicited by these partial α2-adrenoreceptor agonists, clonidine, and dexmedetomidine, compared with full α2-agonists. Our results support the development of specific full and systemic α2-adrenoreceptor agonists for ticagrelor reversal.

Toxoplasma gondii adenosine kinase: expression, purification, characterization, crystallization and preliminary crystallographic analysis

2000 ◽  
Vol 56 (1) ◽  
pp. 76-78 ◽  
Author(s):  
Rosario Recacha ◽  
Alexander Talalaev ◽  
Lawrence J. DeLucas ◽  
Debasish Chattopadhyay
Keyword(s):  
Toxoplasma Gondii ◽  
Recombinant Protein ◽  
Protozoan Parasite ◽  
Adenosine Monophosphate ◽  
Crystallographic Analysis ◽  
Adenosine Kinase ◽  
Monoclinic Space Group ◽  
Cell Parameters ◽  
Ph Range

The obligate intracellular protozoan parasite Toxoplasma gondii depends on the purine-salvage pathway for its purine supply. Unlike its mammalian hosts, T. gondii salvages purine precursors predominantly via adenosine kinase, the enzyme that phosphorylates adenosine to adenosine monophosphate (AMP). The cDNA encoding T. gondii adenosine kinase was subcloned and expressed in Escherichia coli. The recombinant protein was active in an in vitro enzyme assay over a broad pH range. It required a divalent cation for activity. The enzyme was inactivated by the addition of 1 µM mercuric chloride. The inactivation could be reversed by a reducing agent. The active recombinant protein was crystallized using sodium sulfate as precipitant at pH 8.0. The crystals diffract to 1.8 Å and belong to the monoclinic space group P21, with unit-cell parameters a = 47.5, b = 68.9, c = 57.0 Å, β = 100.3°. The calculated Vm based on one molecule per asymmetric unit is 2.38 Å3 Da−1.

Response of canine cardiocyte lysosomes to ATP

1985 ◽  
Vol 249 (1) ◽  
pp. H20-H28 ◽  
Author(s):  
A. M. Spanier ◽  
B. F. Dickens ◽  
W. B. Weglicki
Keyword(s):  
Cathepsin B ◽  
Ventricular Myocytes ◽  
Adenosine Diphosphate ◽  
Cross Contamination ◽  
Significant Protection ◽  
Inorganic Pyrophosphate ◽  
Relationship Of ◽  
The Relationship

The present study was designed to evaluate the relationship of adenosine triphosphate (ATP) to maintenance of cardiac lysosome latency. Highly latent lysosomes were isolated from adult canine ventricular myocytes or cardiocytes and exhibited latency values (based on % free N-acetyl-beta-glucosaminidase, NAGA) of 29.3 +/- 4.7% (SE), minimal cross contamination by mitochondria (less than 2% cardiolipin by weight) and only 1.68% of the total cytochrome c oxidase activity; enzymatic enrichments ranged from 10-fold for NAGA to almost 50-fold for cathepsin B. Incubations of cardiocyte lysosomes at pH 7.0 and 25 degrees C for up to 1 h resulted in changes in the rate of loss in lysosomal latency when ATP levels were adjusted from 0.0 to 5.0 mM; under these conditions as ED50 of 0.62 mM ATP was determined. The protection afforded by ATP was reversed by addition of the H+ ionophore m-chlorocarbonylcyanide phenylhydrazone (CCCP) at 10 microM to the lysosomal suspensions. A significant increase in lysosomal membrane fluidity, measured by fluorescent polarization of diphenylhexatriene, was also seen in the absence of ATP. Adenosine diphosphate (ADP) afforded significant protection only at the very highest concentration (5.0 mM); inorganic pyrophosphate (PPi) did not protect against loss of latency at any concentration. Thus ATP exerts a significant stabilizing effect on cardio(myo)cyte lysosomes in vitro and may be one of the metabolites regulating lysosomal integrity in vivo.

scholarly journals CFAP45 deficiency causes situs abnormalities and asthenospermia by disrupting an axonemal adenine nucleotide homeostasis module

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Gerard W. Dougherty ◽  
Katsutoshi Mizuno ◽  
Tabea Nöthe-Menchen ◽  
Yayoi Ikawa ◽  
Karsten Boldt ◽  
...  
Keyword(s):  
Atp Hydrolysis ◽  
Adenine Nucleotide ◽  
Adenosine Monophosphate ◽  
Adenosine Diphosphate ◽  
Situs Inversus Totalis ◽  
Proteomic Profiling ◽  
Binding Interface ◽  
Axonemal Dynein ◽  
Cilia And Flagella

Abstract Axonemal dynein ATPases direct ciliary and flagellar beating via adenosine triphosphate (ATP) hydrolysis. The modulatory effect of adenosine monophosphate (AMP) and adenosine diphosphate (ADP) on flagellar beating is not fully understood. Here, we describe a deficiency of cilia and flagella associated protein 45 (CFAP45) in humans and mice that presents a motile ciliopathy featuring situs inversus totalis and asthenospermia. CFAP45-deficient cilia and flagella show normal morphology and axonemal ultrastructure. Proteomic profiling links CFAP45 to an axonemal module including dynein ATPases and adenylate kinase as well as CFAP52, whose mutations cause a similar ciliopathy. CFAP45 binds AMP in vitro, consistent with structural modelling that identifies an AMP-binding interface between CFAP45 and AK8. Microtubule sliding of dyskinetic sperm from Cfap45−/− mice is rescued with the addition of either AMP or ADP with ATP, compared to ATP alone. We propose that CFAP45 supports mammalian ciliary and flagellar beating via an adenine nucleotide homeostasis module.

scholarly journals THE ACQUISITION OF FORWARD MOTILITY IN THE SPERMATOZOA OF THE POLYCHAETE ARENICOLA MARINA

1994 ◽  
Vol 195 (1) ◽  
pp. 259-280
Author(s):  
A Pacey ◽  
J Cosson ◽  
M Bentley
Keyword(s):  
Sperm Motility ◽  
Sea Water ◽  
Beat Frequency ◽  
Flagellar Apparatus ◽  
Pharmacological Agents ◽  
Sperm Activation ◽  
Arenicola Marina ◽  
First Results

Sperm activation in the polychaete annelid Arenicola marina was investigated using video microscopy following the in vitro and in vivo manipulation of gametes. Careful observation of spermatozoa obtained from spawning animals indicated that they were immotile immediately after their release from the gonopores, but that they subsequently became motile following dilution in sea water. It was determined that under the pH conditions of the coelomic cavity (pH 7.3) sperm motility was suppressed, whereas upon dilution in sea water buffered at pH 8.2, motility was triggered. It is hypothesised that sperm activation, under normal conditions, occurs in two stages. The first results in the liberation of free spermatozoa from sperm morulae, allowing them to be spawned; this process is stimulated by an endocrine factor and occurs in vivo during normal spawning. The second involves the switching on of the sperm flagellar apparatus, which occurs when spermatozoa are subjected to change in extracellular pH associated with their dilution in sea water. Pharmacological agents such as ammonium chloride and quinacrine are shown to stimulate the breakdown of sperm morulae and the acquisition of sperm motility. Motile spermatozoa of A. marina, in artificial sea water buffered at pH 8.2, can remain motile for over 5 h, have a beat frequency of approximately 50 Hz and have average path velocities of between 100 and 120 µm s-1. Motile spermatozoa under these conditions can also display the phenomenon of intermittent swimming.

The Rôle of Human Platelets and Plasma in the Metabolism of Adenosine Diphosphate and Monophosphate Added In Vitro

1964 ◽  
Vol 12 (02) ◽  
pp. 510-523
Author(s):  
G. P Kerby ◽  
S. M Taylor
Keyword(s):  
Human Blood ◽  
Adenylate Kinase ◽  
Adenosine Monophosphate ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Human Blood Plasma ◽  
Alkaline Phosphatases ◽  
Adenylic Acid

SummaryWhen exogenous adenosine monophosphate (AMP) and diphosphate (ADP) were added to sonicated platelet-rich human blood plasma in AMP-ADP ratio approximating that normally present in platelets, a ratio close to 1.0 was consistently established within 20 to 60 minutes of incubation at 37° C. This resulted both from progressive disappearance of ADP and from initial augmentation followed by disappearance of AMP, the disappearance of ADP always being greater however than the disappearance of AMP so that the higher ratio was maintained.AMP appeared as ADP disappeared during the first 20 minutes of incubation. Platelet adenylate kinase, plasma 5-adenylic acid deaminase and nonspecific alkaline phosphatases appeared to be of particular importance in achieving the changing ratios of nucleotides.

Ciliary coordination in newt lung cells requires microtubule-based linkages between basal bodies: A correlative light and EM study

1992 ◽  
Vol 50 (1) ◽  
pp. 570-571
Author(s):  
Robert Hard ◽  
Gerald Rupp ◽  
Matthew L. Withiam-Leitch ◽  
Lisa Cardamone
Keyword(s):  
Basal Body ◽  
Untreated Control ◽  
Beat Frequency ◽  
Primary Cultures ◽  
Living Cells ◽  
Power Stroke ◽  
Ultrastructural Changes ◽  
Lung Cells ◽  
Basal Bodies ◽  
Ciliated Cells

In a coordinated field of beating cilia, the direction of the power stroke is correlated with the orientation of basal body appendages, called basal feet. In newt lung ciliated cells, adjacent basal feet are interconnected by cold-stable microtubules (basal MTs). In the present study, we investigate the hypothesis that these basal MTs stabilize ciliary distribution and alignment. To accomplish this, newt lung primary cultures were treated with the microtubule disrupting agent, Colcemid. In newt lung cultures, cilia normally disperse in a characteristic fashion as the mucociliary epithelium migrates from the tissue explant. Four arbitrary, but progressive stages of dispersion were defined and used to monitor this redistribution process. Ciliaiy beat frequency, coordination, and dispersion were assessed for 91 hrs in untreated (control) and treated cultures. When compared to controls, cilia dispersed more rapidly and ciliary coordination decreased markedly in cultures treated with Colcemid (2 mM). Correlative LM/EM was used to assess whether these effects of Colcemid were coupled to ultrastructural changes. Living cells were defined as having coordinated or uncoordinated cilia and then were processed for transmission EM.

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