scholarly journals Studies on the time course and rate-limiting steps in the activation of adenylate cyclase in rat liver by cholera toxin

1978 ◽  
Vol 173 (1) ◽  
pp. 59-64 ◽  
Author(s):  
J Fischer ◽  
T R Kohler ◽  
L G Lipson ◽  
J Flores ◽  
P A Witkum ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Rat Liver ◽  
Time Course ◽  
Intact Cell ◽  
Intact Cells ◽  
Maximal Stimulation ◽  
Rate Limiting

Cholera toxin stimulates adenylate cyclase in rat liver after intravenous injection. The stimulation follows a short latent period of 10min, and maximum stimulation was attained at 120min. Half-maximal stimulation was achieved at 35min. In contrast with this lengthy time course in the intact cell, adenylate cyclase in broken-cell preparations of rat liver in vitro were maximally stimulated by cholera toxin (in the presence of NAD+) in 20min with half-maximal stimulation in 8min. Binding of cholera toxin to cell membranes by the B subunits is followed by translocation of the A subunit into the cell or cell membrane, and separation of the A1 polypeptide chain from the A2 chain by disulphide-bond reduction, and finally activation of adenylate cyclase by the A1 chain and NAD+. As the binding of cholera toxin is rapid, two possible rate-limiting steps could be the determinants of the long time course of action. These are translocation of the A1 chain from the outside of the cell membrane to its site of action (this includes the time required for separation from the whole toxin) or the availability of NAD+ for activation. When NAD+ concentrations in rat liver were elevated 4-fold, by the administration of nicotinamide, no change in the rate of activation of adenylate cyclase by cholera toxin was observed. Thus the intracellular concentration of NAD+ is not rate-limiting and the major rate-limiting determinant in intact cells must be between the time of toxin binding to the cell membrane and the appearance of subunit A1 at the enzyme site.

scholarly journals Activation by cholera toxin of adenylate cyclase solubilized from rat liver

1976 ◽  
Vol 157 (3) ◽  
pp. 785-787 ◽  
Author(s):  
S Van Heyningen
Keyword(s):  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Rat Liver ◽  
Thiol Group ◽  
Subunit B

Cholera toxin, or peptide A1 from the toxin, activates adenylate cyclase solubilized from rat liver with Lubrol PX, provided that cell sap, NAD+, ATP and thiol-group-containing compounds are present. The activation is abolished by antisera to whole toxin, but not to subunit B.

F-actin serves as a template for cytokeratin organization in cell free extracts

2002 ◽  
Vol 115 (7) ◽  
pp. 1373-1382 ◽  
Author(s):  
Kari L. Weber ◽  
William M. Bement
Keyword(s):  
Time Course ◽  
Early Time ◽  
Dynamic Imaging ◽  
Actin Network ◽  
Intact Cells ◽  
Time Points ◽  
Egg Extracts ◽  
Actin Cables

The microtubule, F-actin, and intermediate filament systems are often studied as isolated systems, yet the three display mutual interdependence in living cells. To overcome limitations inherent in analysis of polymer-polymer interactions in intact cells, associations between these systems were assessed in Xenopus egg extracts. In both fixed and unfixed extract preparations, cytokeratin associated with F-actin cables that spontaneously assembled in the extracts. Time-course experiments revealed that at early time points cytokeratin cables were invariably associated with F-actin cables,while at later time points they could be found without associated F-actin. In extract samples where F-actin assembly was prevented, cytokeratin formed unorganized aggregates rather than cables. Dynamic imaging revealed transport of cytokeratin by moving F-actin as well as examples of cytokeratin release from F-actin. Experimental alteration of F-actin network organization by addition of α-actinin resulted in a corresponding change in the organization of the cytokeratin network. Finally, pharmacological disruption of the F-actin network in intact, activated eggs disrupted the normal pattern of cytokeratin assembly. These results provide direct evidence for an association between F-actin and cytokeratin in vitro and in vivo, and indicate that this interaction is necessary for proper cytokeratin assembly after transition into the first mitotic interphase of Xenopus.

Reversible effects of phenothiazines on frog gastric stimulation

1984 ◽  
Vol 247 (4) ◽  
pp. G366-G376
Author(s):  
N. Raphael ◽  
E. B. Ekblad ◽  
T. E. Machen
Keyword(s):  
Adenylate Cyclase ◽  
Time Course ◽  
Phosphodiesterase Inhibitor ◽  
Secretory Activity ◽  
Gastric Stimulation ◽  
Camp Content ◽  
Oxyntic Cell ◽  
Camp Generation ◽  
Stimulus Secretion Coupling

The calmodulin inhibitors trifluoperazine (TFP), chlorpromazine (CPZ), and promethazine (PZ) were tested for effects on stimulus-secretion coupling in in vitro bullfrog gastric mucosa. When added to histamine-stimulated tissues, the drugs caused H+ secretion to decrease and transepithelial resistance to increase over a 2-h time course. The potency sequence was TFP (IC50 = 40 microM) greater than CPZ (IC50 = 72 microM) congruent to PZ (IC50 = 72 microM). Anesthetics and other phenothiazines with weak anticalmodulin activity had no effect on secretory parameters. In the presence of histamine, further addition of isobutylmethylxanthine (IBMX, a phosphodiesterase inhibitor) plus dibutyryl cAMP (DBcAMP), IBMX alone, or forskolin (a specific activator of adenylate cyclase) to phenothiazine-inhibited tissues caused full resumption of secretory activity. If TFP (50 microM) was added before stimulation with histamine, the normal increases in tissue cAMP content (which occurs primarily in oxyntic cells), oxyntic cell apical membrane elaboration (morphometric analysis of electron micrographs), and H+ secretion were all blocked. Subsequent addition of IBMX or IBMX plus DBcAMP completely reversed the TFP effect. These results indicate that the histamine-sensitive adenylate cyclase may be the site of TFP inhibition and Ca2+-calmodulin regulation; since these drugs inhibited stimulation by DBcAMP plus IBMX, they may also be exerting additional effects distal to cAMP generation.

Regulation of the purine salvage pathway in rat liver

1992 ◽  
Vol 262 (3) ◽  
pp. E344-E352 ◽  
Author(s):  
Y. A. Kim ◽  
M. T. King ◽  
W. E. Teague ◽  
G. A. Rufo ◽  
R. L. Veech ◽  
...  
Keyword(s):  
Rat Liver ◽  
De Novo ◽  
Equilibrium Constants ◽  
Purine Metabolism ◽  
Purine Salvage ◽  
Rate Limiting Step ◽  
Delta G ◽  
Salvage Pathways ◽  
Rate Limiting

The regulation of purine metabolism in rat liver has been examined under conditions that alter the flux through the pathway. Rats were given intraperitoneal injections of ethanol, sodium acetate, or sodium phosphate to attain body water concentrations of approximately 70, 20, and 10 mM, respectively. The livers were freeze-clamped after 30 min, and extracts were made for the analysis of metabolites, cofactors, purine bases, and nucleosides; homogenates were made for the measurement of the activities and kinetic parameters of seven enzymes that participate in purine salvage. The values of the equilibrium constants of nine reactions were determined in vitro and compared with the ratios of the reactants measured in liver. The changes in phosphoribosylpyrophosphate (PRPP), a key intermediate in both the de novo and salvage pathways of purine metabolism, were directly correlated with the changes in ribose 5-phosphate (ribose-5-P); ([PRPP] = 1.7[ribose-5-P] - 7.4 mumol/kg). Ribose-5-P concentrations in turn could be predicted from the liver content of fructose 6-phosphate and glyceraldehyde 3-phosphate by calculation from the known equilibria. The maximum velocities in the tissue of the seven enzymes measured were calculated from the measured substrate values in the liver and with consideration of other effectors of enzyme activity. PRPP synthetase was the least active of the enzymes measured, indicating a possible rate-limiting step. The delta G of the enzyme steps differed from equilibrium values by factors ranging from 4 (nucleoside phosphorylase) to 10(5) (PRPP synthetase and purine transferase reactions). The regulation of purine salvage appeared to depend on the levels of PRPP and ribose-5-P.

Molecular and cellular requirements for the regulation of adenylate cyclases by calcium

2003 ◽  
Vol 31 (5) ◽  
pp. 912-915 ◽  
Author(s):  
D.M.F. Cooper
Keyword(s):  
Adenylate Cyclase ◽  
Protein Interactions ◽  
Molecular Mechanisms ◽  
Intact Cell ◽  
Protein Protein Interactions ◽  
Additional Layer ◽  
Additional Protein ◽  
Signalling Systems

Calcium-sensitive adenylate cyclases provide a key regulatory device for integrating the activities of the two major signalling systems, Ca2+ and cAMP. Recent experiments have brought us closer to understanding the molecular mechanisms whereby Ca2+ either stimulates or inhibits susceptible adenylate cyclases in vitro. However, in the intact cell an additional layer of sophistication is evident whereby Ca2+-sensitive adenylate cyclases are juxtaposed with Ca2+-entry channels, such that the cyclases respond selectively to capacitative Ca2+ entry. Part of this dependency is enforced by the placement of Ca2+-sensitive adenylate cyclases (AC5, AC6 and AC8) in caveolae, from which at least one Ca2+-insensitive adenylate cyclase (AC7) is excluded. However, additional protein–protein interactions are also required to ensure the dependency of these cyclases on capacitative Ca2+ entry. Recent findings in this area and their implications for ‘local cAMP signals’ will be discussed.

Assessment of changes in sperm cell membrane integrity occurring during the storage of semen from genetically different males using two diagnostic methods

2014 ◽  
Vol 94 (4) ◽  
pp. 601-606 ◽  
Author(s):  
Anna Wysokińska ◽  
Stanislaw Kondracki
Keyword(s):  
Cell Membrane ◽  
Sperm Cell ◽  
Cell Membranes ◽  
Intact Cell ◽  
Membrane Integrity ◽  
Diagnostic Methods ◽  
Cell Membrane Integrity ◽  
Staining Methods ◽  
Breed Crosses

Wysokińska, A. and Kondracki, S. 2014. Assessment of changes in sperm cell membrane integrity occurring during the storage of semen from genetically different males using two diagnostic methods. Can. J. Anim. Sci. 94: 601–606. The present study was carried out to assess changes in sperm cell membrane integrity occurring during the storage of semen collected from genetically different domestic male pigs. The study was aimed at assessing differences in the course of changes in the integrity of cell membranes in spermatozoa produced by males with different degrees of genetic diversity (pure-bred males, two-breed hybrids and multi-breed crosses) and testing the usefulness of two methods of sperm cell membrane integrity evaluation, based on material collected from genetically different males. The experiments were conducted on 56 ejaculates collected from 28 domestic male pigs. The examination of sperm cell membrane integrity was performed three times for each ejaculate, i.e., after 1 h, after 24 h and after 48 h from collection. The preparations for analysing cell membrane integrity were made using two methods: the SYBR 14/PI method and the eosin–nigrosin method. It was found that both SYBR 14/PI and eosin–nigrosin staining methods make it possible to successfully assess the integrity of the plasma membrane of domestic pig sperm cells under in vitro conditions. Hybrid pig spermatozoa, especially those from multi-breed crosses, better retain the integrity of their plasmalemmas than the spermatozoa of pure-bred boars. The ejaculates of Hypor cross-breed boars assessed after 1, 24 and 48 h of storage contain more spermatozoa with intact cell membranes than the ejaculates of pure-bred Duroc and Pietrain boars. The ejaculates of Hypor boars also show fewer decaying spermatozoa than those produced by pure-bred boars.

scholarly journals The Application of PMA (Propidium Monoazide) to Different Target Sequence Lengths of Zebrafish eDNA: A New Approach Aimed Toward Improving Environmental DNA Ecology and Biological Surveillance

2021 ◽  
Vol 9 ◽  
Author(s):  
Takaya Hirohara ◽  
Kenji Tsuri ◽  
Koichi Miyagawa ◽  
Robert T. R. Paine ◽  
Hiroki Yamanaka
Keyword(s):  
Cell Membrane ◽  
Intact Cell ◽  
Covalent Bond ◽  
Environmental Dna ◽  
Sequence Length ◽  
Target Sequence ◽  
Type I ◽  
Propidium Monoazide ◽  
Intact Cells ◽  
Double Stranded Dna

Environmental DNA (eDNA) analysis has enabled more sensitive and efficient biological monitoring than traditional methods. However, since the target species is not directly observed, interpretation of results cannot preclude process Type I errors. Specifically, there may be a spatial or temporal gap between the target eDNA and the eDNA source in the sampled area. Moreover, eDNA surveillance lacks the ability to distinguish whether eDNA originated from a living or non-living source. This kind of Type I error is difficult to control for, in part, because the relationship between the state of eDNA (i.e., intracellular or extracellular) and the degradation rate is still unclear. Here, we applied PMA (Propidium monoazide) to eDNA analysis which enabled us to differentiate “intact cells” from “disrupted cells.” PMA is a dye that has a high affinity for double-stranded DNA and forms a covalent bond with double-stranded DNA and inhibits amplification of the bonded DNA molecules by PCR. Since PMA is impermeable to the cell membrane, DNA protected by an intact cell membrane can be selectively detected. In this study, we investigated the workability of PMA on vertebrate eDNA using zebrafish, Danio rerio. Aquarium water was incubated for 1 week to monitor the eDNA degradation process of both intracellular and extracellular eDNA. We developed ten species-specific quantitative PCR assays for D. rerio with different amplification lengths that enabled independent quantification of total eDNA (sum of intracellular and extracellular eDNA, commonly measured in other studies) and intracellular eDNA (DNA in intact cells) and allow for analyses of sequence length-dependent eDNA degradation in combination with PMA. We confirmed that PMA is effective at differentiating “intact” and “disrupted” fish cells. We found that total eDNA and intracellular eDNA have different degradation processes that are dependent on the length of target sequence. For future conservation efforts using eDNA analyses, it is necessary to increase the reliability of the analysis results. The research presented here provides new analysis tools that expand our understanding of the ecology of eDNA, so that more accurate and reliable conclusions can be determined.

MODULATION OF CYCLIC AMP IN ISOLATED RAT UTERINE TISSUE SLICES BY PORCINE RELAXIN

1980 ◽  
Vol 87 (1) ◽  
pp. 153-159 ◽  
Author(s):  
D. G. JUDSON ◽  
SARAH PAY ◽  
K. D. BHOOLA
Keyword(s):  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Time Course ◽  
Tissue Concentration ◽  
Uterine Tissue ◽  
Tissue Slices ◽  
Rat Uterus ◽  
Hormonal Action ◽  
Isolated Rat

Porcine relaxin produced a rapid, dose-related rise of cyclic AMP values in rat uterine tissue incubated in vitro. In time-course experiments, peak cyclic AMP concentrations were observed in the uterine slices at 5 min; subsequently the values fell, at first rapidly and then more slowly with the tissue concentration remaining significantly raised at 15 min. Levels of cyclic GMP in the same tissue slices were not significantly altered by relaxin. Furthermore, no increase in basal cyclic AMP values was measured in control slices prepared from the rat heart or jejunum. An increase in cyclic AMP concentration comparable to that found in the rat uterus was observed in slices of porcine uterus and cervix but not of vagina when they were stimulated with porcine relaxin. Our results suggest that the hormonal action of relaxin on the uterus and cervix is mediated through receptors linked to the enzyme, adenylate cyclase.

scholarly journals Human platelets are defective in processing of cholera toxin

1983 ◽  
Vol 212 (3) ◽  
pp. 669-678 ◽  
Author(s):  
R J Hughes ◽  
P A Insel
Keyword(s):  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Mammalian Cells ◽  
Time Course ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Human Platelets ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity

Cholera toxin is unable to elevate cyclic AMP levels in intact human platelets despite being very efficacious in this respect in other mammalian cells; in the presence of 0.5 mM-isobutylmethylxanthine, we found that 3-6nM-cholera toxin over 3h at 37 degrees C elevated platelet cyclic AMP from 33 +/- 13 to 39 +/- 12pmol/mg of protein (means +/- S.D.; n = 12). We have investigated the basis for this lack of response. 125I-labelled cholera toxin bound to platelets both saturably and with high affinity (Kd congruent to 60pM; Bmax. congruent to 50fmol/mg of protein). Incubation of platelets with the putative cholera toxin receptor monosialoganglioside GM1 enhanced 125I-labelled cholera toxin binding at least 40-fold but facilitated only a minimal (less than or equal to 3-fold) elevation of platelet cyclic AMP levels. In contrast, dithiothreitol-activated cholera toxin markedly stimulated adenylate cyclase activity in platelet membranes. Platelet cytosol both enhanced stimulation of adenylate cyclase activity by activated cholera toxin (A1 subunit) and supported stimulation by the A1-A2 subunit of cholera toxin. Neither GTP nor NAD+, both necessary for response to cholera toxin, was lacking in intact platelets. However, we found that platelets were unable to cleave cholera toxin to the active A1 subunit (as assessed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis). By contrast, murine S49 lymphoma cells were able to generate the A1 subunit with a time course that closely resembled the kinetics of toxin-mediated cyclic AMP accumulation in these cells. Thus we conclude that human platelets are defective in their ability to process surface-bound cholera toxin. These results indicate that binding of cholera toxin to surface receptors is necessary, but not sufficient, for expression of the toxin effect and the generation of the A1 subunit of the toxin may be rate-limiting for expression of cholera toxin response.

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