Concentration-activity Curves Research Articles

Test–Retest Reproducibility of [11C]-(+)-Propyl-Hexahydro-Naphtho-Oxazin Positron Emission Tomography Using the Bolus plus Constant Infusion Paradigm

We examined the reproducibility of using the constant infusion paradigm for equilibrium measurement of D2/3 receptors using [11C]-(+)-propyl-hexahydro-naphtho-oxazin (PHNO) positron emission tomography (PET). Six subjects were scanned with a bolus plus constant infusion (Kbol = 80 minutes) of [11C]-(+)-PHNO. Binding potential (BPND) was computed using the equilibrium approach and compared to a simplified reference tissue model (SRTM). The rate of change in the concentration-activity curve from 60 to 90 minutes was -5 ± 13%/h in the caudate, putamen, substantia nigra, thalamus, and cerebellum but was 15 ± 15%/h in the ventral striatum and pallidum. Test-retest variability was lower in striatal compared to extrastriatal regions (4 ± 8% vs -8 ± 22%, respectively) using the equilibrium approach, with comparable results with SRTM. The equilibrium ratio and SRTM yielded reliable BPND estimates (intraclass correlation coefficient = 0.88 and 0.82, respectively). These studies support the reproducibility of the bolus plus constant infusion paradigm with [11C]-(+)-PHNO PET.

Allosteric Transinhibition by Specific Antagonists in CCR2/CXCR4 Heterodimers

Chemokine receptors are presently used as targets for candidate drugs in the frame of inflammatory diseases and human immunodeficiency virus infection. They were shown to dimerize, but the functional relevance of dimerization in terms of drug action remains poorly understood. We reported previously the existence of negative binding cooperativity between the subunits of CCR2/CCR5 heterodimers. In the present study, we extend these observations to heterodimers formed by CCR2 and CXCR4, which are more distantly related. We also show that specific antagonists of one receptor inhibit the binding of chemokines to the other receptor as a consequence of their heterodimerization, both in recombinant cell lines and primary leukocytes. This resulted in a significant functional cross-inhibition in terms of calcium mobilization and chemotaxis. These data demonstrate that chemokine receptor antagonists regulate allosterically the functional properties of receptors on which they do not bind directly, with important implications on the effects of these potential therapeutic agents.

Cytochrome P4501A induction potencies of polycyclic aromatic hydrocarbons in a fish hepatoma cell line: Demonstration of additive interactions

AbstractThe relative CYP1A induction potencies, determined as ethoxyresorufin‐O‐deethylase (EROD) activity, and the cytotoxicities of 19 compounds with one to six benzene rings, mixtures of polycyclic aromatic hydrocarbons (PAHs), and contaminated landfill leachates have been determined in the permanent fish hepatoma cell line PLHC‐1. No CYP1A induction was observed with benzene, naphthalene, anthracene, acenaphthene, benzo[g,h,i]perylene, and fluorene and low induction was found with fluoranthene and phenanthrene. All other PAHs with three and more benzene rings led to a concentration‐related induction of CYP1A, with rebound decreases at high concentrations resulting in bell‐shaped concentration–activity curves. Fish‐related induction equivalency factors (IEFs) were estimated for all PAHs on the basis of EC50 values of their EROD activities and are reported here for the first time. The following order of decreasing IEFs was found: dibenz[a,h]anthracene > dibenzo[a,i]pyrene > benzo[k]fluoranthene > 3‐methylcholanthrene > benzo[a]pyrene > benzo[e]pyrene > chrysene > 7,12‐dimethylbenz[a]anthracene > perylene > benz[a]anthracene > pyrene. In contrast to the EROD activity, the immunodetectable protein content determined by ELISA showed a concentration‐dependent increase. The interaction of PAHs in mixtures of up to eight individual compounds was additive based on their EROD activities. In landfill leachates, determined induction equivalents (IEQ) were significantly higher than calculated IEQs based on analytical measurements, which indicates additional unknown inducing compounds present in leachates. This study shows that the PLHC‐1 cell in vitro system serves as an integrative bioanalytical tool in the ecotoxicological evaluation of aquatic environmental samples contaminated with CYP1A‐inducing compounds.

Preselection of Potential Cancerostatics by Automatic Analysis of Suspended and Adherent Cells Incubated in Microplates

Alternative toxicological screening programmes, without the use of animal experiments, are intended to eliminate dangerous substances and to find new pharmacologically active agents in cell cultures. They can also provide information on the cytostatic activities of the agents. Intercalating cytostatics which bind DNA were selected by measuring the statistical distributions of the cell diameters of K-562 and L-929 cells by using an electronic cell analyser (CASY1). These compounds were identified by cell enlargement or from flat concentration-activity curves created with the cell analyser system. Incubation for 72 hours with DNA-binding agents, such as doxorubicin, daunorubicin and Mitoxantron®, resulted in enlargement of cell diameter and cell volume. The antineoplastic agents actinomycin D and ambazone had no comparable effect. Comparisons of the different parameters obtained with CASY1 measurement were performed with Microsoft EXCEL.

Cellulase activity of soils

Cellulases are a group of enzymes that catalyze the degradation of cellulose, a polysaccharide built of β-1,4 linked glucose units. This group consists of endo-1,4-glucanase (EC 3.2.1.4), exo-1,4-glucanase (EC 3.2.1.91), and β-d-glucosidase (EC 3.2.1.21). The products of cellulose degradation are glucose, cellobiose, and higher molecular weight oligosaccharides. An accurate and precise method for the assay of cellulase activity in soil was developed. It involves determination of the reducing sugars produced when a soil sample is incubated with acetate buffer (50 mM, pH 5.5), carboxymethyl cellulose (CMC), and toluene at 30°C for 24 h. Results showed that the optimal pH of cellulase activity was 5.5. Reducing sugars produced from cellulase activity increased linearly up to 7 days, and they increased with increasing amounts of soil up to 7 g. The substrate concentration-activity curves of cellulase activity in soils obeyed the Michaelis-Menten equation. In two soils tested, the Michaelis constant (Km) ranged from 9.7 to 21.4 g CMC 1−1. Cellulase activity in soils was maximal at temperatures ranging from 50 to 60°C. The activation energy values of the reaction catalyzed by cellulase in four soils ranged from 21.7 to 28.0 kJ mol−1. Soil cellulase was denatured at temperatures ranging from 60 to 70°C; from 60 to 80%of total cellulase activity remained after heating air-dried soil samples at 100°C for 2 h. The corresponding values for field-moist soils were 18–23%. Toluene treatment markedly increased the cellulase activity values over the nontreated samples. Reducing sugar values increased significantly when air-dried or field-moist soil samples were incubated as described but without CMC, suggesting a role for cellulase in degradation of the native substrates.

Evidence that a form of ATP uncomplexed with divalent cations is the ligand of P2y and nucleotide/P2u receptors on aortic endothelial cells

1. The response of bovine aortic endothelial cells to adenosine 5'-triphosphate (ATP) is mediated by both P2y and nucleotide/P2u receptors. In order to determine which form of the nucleotide is the true ligand of these receptors, we have investigated the effects of divalent cations on ATP-, uridine 5'-triphosphate (UTP)- and 2 methylthioadenosine 5'-triphosphate (2MeSATP)-induced inositol phosphate accumulation in these cells. 2. Omisson of Mg2+ from a calcium-free incubation buffer caused a shift to the left of the ATP concentration-action curve. 3. In the presence of EDTA (1 mM), the basal level of inositol trisphosphate (InsP3) was markedly increased and the absolute maximal response to ATP was decreased; however, the response to low concentrations of ATP was enhanced. 4. When the results were plotted in terms of calculated ATP4- concentrations, the concentration-response curves obtained in the presence of 1.25 mM Mg2+ lay closer to the respective curves obtained when Mg2+ was omitted from the medium or when Mg2+ was omitted and EDTA (1 mM) was added. The curves became almost superimposable when the baseline value was subtracted. 5. A similar shift to the left of the concentrations-action curves was also observed with both UTP and 2MeSATP. 6. Our data provide evidence that a form of ATP uncomplexed with divalent cation is the preferential agonist of both the nucleotide/P2u and the P2y receptors expressed on bovine aortic endothelial cells.

Activity and Partial Purification of Liver Tryptophan Pyrrolase in Rainbow Trout.

Compounds that activate tryptophan pyrrolase in other animals like methylene blue, hematin, EDTA, and calciumion did not affect fish enzyme activity. Neutralized ascorbic acid showed an inhibitory effect. Time-activity and enzyme concentration-activity curves were determined in three methods of assay. Km was estimated via the Lineweaver-Burk plot to be 80mM. The enzyme was partially purified by a four-step process: solubilization, DEAE Sephadex A-50 chromatography (batch method), and by two consecutive DEAE Sepharose CL-6B chromatographs. The partially purified enzyme was highly unstable so that no characterization was at all possible.

Anti-leukotriene D 4 action of a new anti-asthmatic drug (KC-404) on the guinea-pig isolated trachea

1. 1. Anti-asthmatic drug, KC-404, inhibited specifically spontaneous contraction and leukotriene (LT) D 4-induced contraction of guinea-pig trachea. 2. 2. Relaxation of spontaneous contraction by KC-404 was reduced by the treatment of the guinea-pig trachea with a cyclooxygenase inhibitor, flurbiprofen. 3. 3. Some parallel shift of the concentration-action curve of LTD 4 by KC-404 was still observed in the presence of flurbiprofen. 4. 4. Specific binding of [ 3H]LTD 4 to the microsomal fraction of guinea-pig trachea was partially inhibited by KC-404. 5. 5. These results suggest that the relaxing action of trachea of KC-404 is due to the interference of interaction between LTD 4 and its receptor and to the product(s) via cyclooxygenase pathway.

Effects of retinylacetate on the kinetics of rat liver guanylate cyclase: possible interaction with sulfhydryl groups.

The effect of retinylacetate (RA) was investigated on rat liver guanylate cyclase stimulated by nitroprusside (NP). The stimulated enzyme seemed to adhere to Michaëlis-Menten kinetics with an apparent Km for MnGTP of 0.10 mM and a Vmax of 410 pmol cGMP/min. X mg prot. RA (0.1-1mM) dose-dependently inhibited the enzyme stimulated by NP (0.1-10 mM). In the presence of 1 mM RA the activity was only 10% of the control activity. The inhibitory action of RA seemed to be non-competitive since it depressed Vmax without affecting the Km for MnGTP. In contrast, however, RA was instead found to stimulate the enzyme when low concentrations of NP (0.01-0.1 mM) were used. The concentration-activity curve for NP was bell-shaped showing an optimum at 0.5 mM. The inhibition induced by RA could not be surmounted by increasing the NP concentration indicating that RA did not compete with NP. A bell-shaped activity curve was also seen when the enzyme activity was measured in the presence of increasing Mn2+ concentrations and during these conditions RA also caused inhibition. In the presence of the sulphydryl reductant dithiothreitol (DTT), the NP concentration needed for optimal enzyme activation was about 100-fold less than in the absence of DTT. The maximal enzyme activity was also slightly increased. In the presence of DTT, RA was much less effective to induce inhibition of the stimulated enzyme, than when DDT was absent. (In the presence of DTT 1 mM RA caused only 30% inhibition compared to 90% inhibition in its absence).(ABSTRACT TRUNCATED AT 250 WORDS)

A possible mechanism of action of a beta-adrenergic partial agonist (carteolol) in guinea pig taenia caecum.

Carteolol, 5-(3-tert-butylamino-2-hydroxy)propoxy-3, 4-dihydrocarbostyril hydrochloride was found to be a beta-adrenergic partial agonist in the taenia caecum of guinea pig. Concentration response curves of carteolol and isoprenaline were parallelly shifted by propranolol (10(-7) M) suggesting that site of action of both the drugs was the same beta-adrenoceptor. The pD2-values of carteolol obtained from the concentration action curves in mechanical response and increase of tissue concentration of cyclic AMP were significantly different from its pA2-value against isoprenaline and its pKi-value (negative log of its apparent dissociation constant). A possible explanation of our observation would seem to be as follows. Carteolol and isopranaline interact with the same beta-adrenoceptor where there may be two different sites: a binding site for agonist and binding site for antagonist.

DOPAMINE RECEPTOR IN ANTERIOR BYSSUS RETRACTOR MUSCLE OF MYTILUS EDULIS

AbstractEffects of dopamine, N-methyl-, ethyl- and propyl-derivatives of dopamine, and alpha- and beta-adrenoceptor stimulants on catch contraction of anterior byssus retractor muscle of Mytilus edulis were tested. The test drugs except the beta-adrenoceptor stimulants relaxed catch contraction. Dopamine was most active and substitution of amino group in dopamine with ethyl and propyl decreased activity considerably. The concentration-action curves of dopamine, its derivatives and norepinephrine shifted in parallel with application of haloperidol but were not influenced by the alpha- and beta-adrenoceptor antagonists. These results suggest that relaxation of catch contraction by catecholamines is mediated through a dopamine receptor. This muscle is considered to be suitable for a study of the dopamine receptor.

Rat Liver Glutamine 5-Phosphoribosyl-1-Pyrophosphate Amidotransferase [EC 2.4.2.14

Glutamine 5-phosphoribosyl-1-pyrophosphate (PRPP) amidotransferase (amidophosphoribosyltransferase), [EC 2.4.2.14] was purified 1,600-fold from rat liver. The preparation gave two protein bands on acrylamide gel electrophoresis, of which only the main band showed enzyme activity. The molecular weight of the enzyme was estimated to be 215,000, 200,000, and 195,000 by Sephadex G-150 gel filtration, polyacrylamide gel electrophoresis, and sucrose density grandient ultracentrifugation, respectively. The apparent Km values for glutamine and PRPP were 1.24 mM and 0.57 mM, respectively. The concentration-activity curve for PRPP changed from a hyperbolic to a sigmoidal form on addition of AMP or GMP, and this inhibition by AMP was prevented by increasing the PRPP concentration. In the presence of high concentrations of inorganic phosphate, the catalytic activity was decreased and the sensitivity to AMP inhibition was slightly increased. The molecular size of liver amidotransferase was not changed by the addition of PRPP, AMP, or 2-mercaptoethanol. The purified rat liver enzyme has a broad pH-range of activity between 6.5 and 8.5.

Microdetermination of inorganic sulfate using thin-layer plates

Inorganic sulfate was precipitated on cellulose thin-layer plates with the radioactive reagent, 133BaCl 2. Excess reagent was removed by repeated washings with an acidic BaCl 2 solution. The residual activity was transferred to vials by cutting out the point of application and its immediate surroundings. Counting was performed in a scintillation well γ-counting system. The concentration-activity curve was linear.

ラット胃幽門洞条片におけるK, Acetylcholine, Baの収縮機序およびCd, Mnの鎮痙機序,とくにCaとの関連

Action mechanisms of the contractile agents, K ACh and Ba and of the antispasmodics, Cd and Mn were investigated. The contractions by K,ACh and Ba are exponential in shape, but consisted of phasic contraction (PC) and the subsequent tonic contraction (TC). PC by K and ACh are inititated by the release and the passive influx of Ca, whereas the PC by BA is due to the release of Ca. On the other hand, TC by these three agents is maintained by the active influx of Ca requiring energy. Since only the contraction by Ba remains constant in Ca(-) bath solution, it is assumed that the direct stimulation to muscle contractile elements without the mediation of Ca mobilization is also partly related to the contracting mechanism of Ba. Storage sites of Ca in the cell membrane of this preparation are distinguished into three divisions, the first, the second and the third, which contains loosely-, less lossely-, and tightly-bound Ca, respectively. K releases Ca to elicit contraction from the first divisions, ACh does so from the first and the second divisions, and Ba does so from all of the three divisions. The following assumption was obtained on the antispasmodic action of Cd and Mn, on the basis of the influence of Ca removed from bath solution and of addition of high K to bath solution and the analysis with concentration-action curves. The antispasmodic mechanism Cd and Mn is due to inhibition of cell membrane (competitive inhibition of influx and then release of Ca and subsequently competitive and non-competitive inhibition of influx and release of Ca) followed by the non-competitive inhibition of muscle contractile system, with the increase of dosage.

モルモット摘出回腸におけるDibenamineのトーヌスに対する作用機序および薬物収縮抑制機序,とくにCaとの関連

Dibenamine (DB) produced contraction due to influx and release of Ca in normal medium, whereas it produced relaxation of the K-induced contraction due to depression of the activity of the muscle cell membrane. DB inhibited active influx, passive influx and release of Ca induced by ACh in this order as the concentrations were increased and also inhibited the contraction by histamine selectively as compared with the contractions by ACh, K and Ba, the inhibition of the ACh-, K- and Ba-contractions being almost to the same degree. In addition, DB inhibited to much the same degree the phasic contraction(PC) and tonic contraction(TC) by histamine, whereas it inhibited TC in preference to PC induced by ACh, K and Ba. Irreversible inhibition by DB of ACh-, K- and Ba-induced contractions were protected by Ca, whereas those of histamine-induced contraction were selectively protected by histamine and antihistamine, but not by Ca. These results indicate that the antagonism of DB and its irreversibility against histamine may be due to blockade of the histaminergic receptor, whereas those against ACh, K and Ba may be due to inhibition of the Ca-site. Evidence has been obtained suggesting that the irreversible parallel shift to the right of the log concentration-action curve of histamine after washout of DB may be due to spare receptors, whereas that of ACh, K or Ba may be due to inhibition of the Ca-site.

Photoaffinity labelling of the β-adrenergic receptors and receptor reserve for isoprenaline

The beta-adrenergic receptors in the taenia isolated from the guinea pig caecum were irreversibly inactivated by photoaffinity labeling with l-isoprenaline and 2-(2-hydroxy-3-isopropylaminopropoxy)iodobenzene. The photoinactivation was elimination in the presence of protectors of the receptors. Photoinactivation caused a considerable parallel shift in the concentration-action curve of l-isoprenaline. The results possibly point to a certain receptor reserve for l-isoprenaline.

Action mechanisms of the contracting drugs, K, acetylcholine, histamine and Ba and of the antispasmodics, isoproterenol and papaverine in the isolated guinea pig ileum, particularly in relation to Ca

Shapes of the contractions induced by K, acetylcholine (AHc), histamine and Ba consisted of the phasic contraction (PC) and the subsequent tonic contraction (TC). PCs by K, ACh and histamine are initiated by the release and the passive influx of Ca, whereas that by Ba is only initiated by the release of Ca. TCs by K, ACh and histamine are maintained by the active influx of Ca, whereas that by Ba is maintained by the active influx and the release of Ca. Storage sites of Ca in the cell membrane of this preparation can be divided into three; the first, the second and the third, which contain the loosely, the less loosely-, and the tightly-bound Ca, respectively. K releases Ca to elicit contraction from the first division, ACh or histamine does so from the first and second divisions, and Ba does so from all of the three divisions. Based on the influence of high K-depolarizing bath solution on the relaxations by isoproterenol (Iso) and papaverine (Pap) and the effects of Iso and Pap on the shapes of contractions by K, ACh, Ba and exogenous Ca, the following assumptions were made: antispasmodic action of Iso is produced by inhibition of cell membrane (inhibition of release and influx of Ca), whereas that by Pap is due to this inhibition followed by inhibition of the muscle contractile system with the increase of concentrations. The effects of Iso and Pap on the concentration-action curves of the contractions by K, ACh, Ba and exogenous Ca(Table II) suggest that the parallel shift to the right of the curves of K, ACh and Ba is due to the functional antagonism between the antispasmodics and the mobilization of Ca produced by the contracting agents.

Factors influencing duodenal trypsin levels following a standard test meal as a test of pancreatic function.

In this investigation, the measurement of trypsin levels in duodenal juice following a standard test meal (Lundh test) was evaluated as a test of pancreatic function, and a study was made of diseases and other factors which may influence its diagnostic efficiency. The method of trypsin assay, which required only basic laboratory equipment, gave a linear concentration-activity curve, with a threshold at 50 mug of crystalline trypsin per ml. Intestinal juice could be frozen and stored for up to six weeks with no detectable loss of tryptic activity. The normal control values were very similar to those found by other workers and were unaffected by the sex or age of the subject. When used to assess 32 patients in whom the presence or absence of pancreatic disease had been clearly established, the test had a diagnostic success rate of 94%. Retrospective analysis of results from 98 patients showed that trypsin levels were generally grossly reduced in patients with chronic pancreatitis or carcinoma of the head of the pancreas. Trypsin levels were normal in most patients with steatorrhoea not of pancreatic origin. Levels were generally depressed to intermediate levels in patients with a diabetic glucose tolerance, with or without steatorrhoea, but no other sign of pancreatic insufficiency. Some lowering of trypsin levels was also noted in patients having an obstruction of the common bile duct. A small but significant depression of tryptic activity was noted in patients with villous atrophy and no pancreatic disease.

Aminoglycosides: biologic effects of molecular manipulation.

Journal Article Aminoglycosides: Biologic Effects of Molecular Manipulation Get access J. Davies, J. Davies Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706 Search for other works by this author on: Oxford Academic PubMed Google Scholar R. Benveniste, R. Benveniste Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706 Search for other works by this author on: Oxford Academic PubMed Google Scholar K. Kvitek, K. Kvitek Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706 Search for other works by this author on: Oxford Academic PubMed Google Scholar B. Ozanne, B. Ozanne Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706 Search for other works by this author on: Oxford Academic PubMed Google Scholar T. Yamada T. Yamada Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706 Search for other works by this author on: Oxford Academic PubMed Google Scholar The Journal of Infectious Diseases, Volume 119, Issue 4-5, April 1969, Pages 351–354, https://doi.org/10.1093/infdis/119.4-5.351 Published: 01 April 1969

A comparison of the properties of the phosphofructokinases of the fat body and flight muscle of the adult male desert locust.

1. Phosphofructokinase was isolated, and partially purified by ammonium sulphate fractionation, from the fat body and flight muscle of the desert locust. 2. Ammonium sulphate appears to stabilize the enzymes, but does not activate them. 3. Both flight-muscle and fat-body enzymes give sigmoidal hexose monophosphate concentration-activity curves, which are characteristic of regulatory enzymes. 4. At low ATP concentrations both the enzyme activities increase rapidly with increasing ATP concentrations, but above an optimum concentration ATP becomes inhibitory. This optimum concentration is 0.2mm for the fat-body enzyme and 0.1mm for the flight-muscle enzyme. 5. AMP activates both the enzymes; half-maximal activation occurs at 10mum in each case, the effect being independent of substrate concentration. 6. 3',5'-(cyclic)-AMP (0.5mm) and P(i) (1mm) activate the flight-muscle enzyme, but have no effect on the fat-body enzyme. 7. FDP (1mm) inhibits both enzymes, and with the flight-muscle enzyme this inhibition is increased by increasing the ATP concentration. 8. Citrate, phosphoenolpyruvate and alpha-glycerophosphate have no effect on either enzyme under the assay conditions used. 9. The properties of phosphofructokinases from the locust are compared with those of phosphofructokinases from other sources.