Target sequences for cis-acting regulation within the dual promoter of the human c-myc gene.

Recombinant plasmids of the human c-myc promoter-leader region and the bacterial chloramphenicol acetyltransferase (cat) gene were constructed. After transfection into different rodent and human cells, the 862-base-pair (bp) PvuII fragment carrying both c-myc promoters and 350 bp of the untranslated leader conferred 1/15 to 1/30 of the CAT activity mediated by the simian virus 40 promoter. The presence of additional sequences upstream of the PvuII fragment had an overall negative effect on c-myc promoter activity detectable by titration analysis with small amounts of transfected plasmid DNA. The analysis of numerous deletion constructs in the c-myc promoter-leader region as well as S1 mapping experiments demonstrated that the high CAT activity depended largely on the presence of the second promoter. By cotransfection of c-myc-cat constructs with plasmids carrying different parts of the c-myc promoter locus, targets for positively acting cellular factors were identified. Two positive regulatory elements were mapped within the 862-bp PvuII fragment. One was localized within the 248-bp PvuII-SmaI fragment -101 to -349 bp upstream of the first cap site and the other within the 142-pb XhoI-NaeI fragment of the first exon, comprising positions -95 to +47 relative to the second cap site. We conclude that the dual promotor of the human c-myc gene represents a strong eucaryotic promotor regulated by cooperation of positively and negatively acting cellular transcription factors.

Anomalies in the polarographic measurement of cytochrome oxidase activity.

Reaction kinetics of the reduction of O2 by cytochrome oxidase follow essentially the same rate equation as that proposed for the oxidation of cytochrome c. However, the apparent second order rate constant varies with the oxidase concentration. The redox level of cytochrome c at the steady state was found to be essentially temperature-independent. Currently recognized pathways (or mechanisms) of electron transport from cytochrome c to O2 do not predict, and cannot account for the occurrence of these phenomena.

Atrial natriuretic peptide receptor modulators: effects of disubstituted quinazolines on receptor binding and in vitro biological activity.

Prazosin (25 microM) was found to increase 125I-labeled rat atrial natriuretic peptide ([125I]rANP) receptor binding by 50% (SC50) in bovine adrenal zona glomerulosa membranes. A series of 2,4-disubstituted quinazolines was prepared in order to identify more potent analogues for additional in vitro testing. Compound 7 (N-[3-[[2-(diethyl-amino)-4-quinazolinyl]amino]propyl] guanidine dinitrate) from this series (3 microM) significantly decreased the EC50 for rANP-mediated inhibition of ACTH-stimulated aldosterone synthesis in rat adrenal glomerulosa cells. At a higher concentration (20 microM), compound 7 had no effect on particulate guanylate cyclase from rabbit glomeruli in either the presence or absence of rANP.

Structural evolution and pharmacology of a novel series of triacid angiotensin II receptor antagonists.

cis-4-(4-Phenoxy)-1-[1-oxo-2(R)-[4-[(2-sulfobenzoyl)amino)-1H- imidazol-1-yl]octyl]-L-proline derivatives represent a novel class of potent nonpeptide angiotensin II (Ang II) receptor antagonists. These compounds evolved from directed structure-activity relationship (SAR) studies on a lead identified by random screening. Further SAR studies revealed that acidic modification of the 4-phenoxy ring system produced a series of triacid derivatives possessing oral activity in pithed rats. The most potent compound, cis-4-[4-(phosphonomethyl)phenoxy]-1-[1-oxo-2(R)-[4-[(2-sulfobenzoyl+ ++) amino]-1H-imidazol-1-yl]octyl]-L-proline (1e), inhibited the pressor response to exogenously administered Ang II for periods up to 8 h following oral dosing. The antihypertensive activity of 1e was evaluated in the Lasix-pretreated conscious spontaneously hypertensive rat (SHR) where it produced a dose-dependent fall in blood pressure following oral dosing lasting > 12 h. Antagonists such as 1e may serve as useful therapeutic agents for the treatment of hypertension as well as for studying the role of Ang II in various disease states.

Saturable adenosine 5'-triphosphate-independent binding of [3H]-ouabain to brain and cardiac tissue in vitro.

1. Several investigators have proposed that membrane Na+, K+-adenosine 5'-triphosphatase (Na+, K+-ATPase) is a mechanism for the transmembrane transport of cardiac glycosides, rather than the receptor for pharmacological actions of these agents. This implies that the glycosides bind to an intracellular constituent (receptor) other than Na+, K+-ATPase. 2. In search for such a receptor site, saturable ATP-independent [3H]-ouabain binding was studied in rat brain and dog and guinea-pig heart homogenates. The binding of the glucoside to this site results in a relatively unstable complex which is stabilized by K+ to a lesser extent than is the complex formed with the ATP-dependent binding to Na+, K+-ATPase. 3. The ATP-independent ouabain binding sites are more abundant in rat brain tissue than in cardiac tissue, and have a lower ouabain affinity compared to the binding sites on Na+, K+-ATPase. 4. These results do not support the contention that there are intracellular inotropic receptors for digitalis.

Differential effect of potassium on the action of digoxin and digoxigenin in guinea-pig heart.

The effect of potassium on the binding of digoxin or digoxigenin to isolated Na+, K+-ATPase was compared with that of potassium on the positive inotropic action of the agents in guinea-pig hearts. The binding of digoxigenin to the enzyme in vitro was reduced to a greater extent by potassium than was the binding of digotoxin. The digoxigenin-induced increase in the force of contraction of left atrial preparations estimated at steady state was reduced at higher potassium concentrations. Potassium had a lesser effect when digoxin was used as the inotropic agent. In contrast, potassium concentrations. Potassium had a lesser effect when digoxin was used as the ininotropic agent. In contrast, potassium reduced the rate of development and also the rate of loss of the positive inotropic action of digoxin observed with left atrial and Langendorff preparations, respectively, to a greater extent than those of digoxigenin. The loss of the positive inotropic effect was more rapid with digoxigenin than with digoxin at each KCl concentration. These data support the contention that the extent of the interaction of digitalis with Na+,K+-ATPase determines the degree of the positive inotropic effect.

3H[2-(2-benzofuranyl)-2-imidazoline] (BFI) binding in human platelets: modulation by tranylcypromine.

2-(2-Benzofuranyl)-2-imidazoline (BFI) is a highly selective ligand for imidazoline-type 2 (I2) binding sites that are known to be associated with monoamine oxidase (MAO). Recently we demonstrated a potentiation of 3H-BFI binding in human but not in rat brain by the nonselective MAO inhibitor tranylcypromine. In the present studies, we evaluated the effect of tranylcypromine on the binding of 3H-BFI to human platelet inner membranes. Membranes were incubated with 3H-BFI at 22 degrees C in 50 mM Tris, 1.5 mM EDTA, pH 7.5. Saturation experiments with 3H-BFI (0.5-80 nM) were analyzed using non-linear curve fitting. Addition of tranylcypromine (0.1 mM) increased the number of 3H-BFI binding sites (Bmax=0.35+/-0.06 vs. 1.87+/-0.15 pmol/mg protein for vehicle and tranylcypromine, respectively) and increased 3H-BFI affinity slightly (KD =16.0+/-4.1 vs. 6.5+/-0.3 nM for vehicle and tranylcypromine, respectively). In competitive binding experiments using the less selective I2 ligand, 3H-idazoxan, tranylcypromine only weakly inhibited binding. Preincubation of platelet membranes with tranylcypromine (1 nM-10 microM) enhanced the Bmax of 3H-BFI binding in a concentration-dependent manner peaking at 1 microM (13 x control) and returning to near baseline at 100 microM. 3H-BFI binding was displaced monophasically (in order of decreasing potency) by BFI > or = 2-(4,5-dihydroimidazol-2-yl)quinoline (BU224) > or = cirazoline >idazoxan >>(1,4-benzodioxan-2-methoxy-2-yl)-2-imidazoline (RX821002)= moxonidine. Amiloride, clorgyline, guanabenz and clonidine displayed biphasic curves with nanomolar high affinity components. Tranylcypromine altered the competition curves for all ligands (except BFI) by increasing the affinities for clonidine and RX821002 and decreasing affinities for BU224, cirazoline, guanabenz, idazoxan, clorgyline, moxonidine, and amiloride. Thus, in human platelets tranylcypromine exposes a high capacity 3H-BFI binding site distinct from previously described I2 sites that retains high affintiy for BFI but not other I2 ligands. Our results suggest that 3H-BFI and 3H-idazoxan may not be considered as interchangeable probes for the I2 binding site.

Reduction of the equilibrium binding of cardiac glycosides and related compounds to Na+,K+-ATPase as a possible mechanism for the potassium-induced reversal of their toxicity.

The influence of potassium ions on the equilibrium state of the binding of cardiac glycosides and their derivatives to partially purified dog heart and rat brain enzyme preparations was studied in vitro. The addition of potassium to the incubation mixture containing enzyme preparation, 3H-ouabain, Na+, Mg2+ and ATP, at the time when the binding reaction is close to equilibrium, caused an immediate reduction of the bound drug concentration; the concentration apparently shifting toward a lower equilibrium state. The degree of the potassium-induced reduction in bound drug concentration was dependent on the potassium concentration and on the chemical structure of the compound. The binding of aglycones, pentacetyl-gitoxin and cassaine was affected to a greater extent than that of the glycosides. These data suggest that one of the mechanisms by which potassium antagonizes the toxic actions of digitalis on the heart is to reduce the drug binding to cardiac Na+,K+-ATPase.

Binding of [3H]2-(2-benzofuranyl)-2-imidazoline (BFI) to human brain: potentiation by tranylcypromine.

Recently, a new imidazoline (I2) ligand, [3H]2-(2-benzofuranyl)-2-imidazoline (BFI) was shown to be more selective for I2 vs alpha 2 binding in rodent brain. We characterized [3H]BFI binding in human brain cortex and lateral reticular nucleus (NRL). Membranes were incubated with [3H]BFI at 22 degrees C in 50 mM Tris, 1.5 mM EDTA at pH 7.5. Saturation experiments with [3H]BFI (0.5-80 nM) were analyzed using non-linear curve fitting. The NRL had 4X more binding sites than cortex with similar affinity (Bmax = 2085 +/- 732 and 471 +/- 41 fmol/mg protein; KD = 9.3 +/- 3.5 and 11.9 +/- 2.7 nM, respectively). In competition studies, cortical [3H]BFI binding was displaced in order of decreasing potency by clorgyline > BFI > or = cirazoline > idazoxan > or = guanabenz > clonidine > RX821002. The monoamine oxidase (MAO) inhibitor tranylcypromine (TCP) (1 nM-10 microM), markedly enhanced [3H]BFI binding in both NRL and cortex. Enhanced binding was maximal at 300 nM (12 X control) and returned to baseline at 30 microM. Potentiation was not seen with pargyline or clorgyline. TCP did not effect [3H]BFI binding in rat cortex, or [3H]idazoxan binding in human cortex and NRL. In human cortex, inhibition of MAO by preincubation with pargyline (10 micro M) abolished the TCP effect. Upon preincubation with TCP, the stimulation of [3H]BFI binding was dose-dependently related to a simultaneous inhibition of MAO. Thus, [3H]BFI labels a site in human NRL and cortex that appears similar to the previously described I2 site labeled by [3H]idazoxan. However, [3H]BFI binding is dramatically stimulated by TCP in human brain via a mechanism dependent on endogenous MAO activity.

Comparison between Aging of Slices and Ethylene Treatment of Whole White Potato Tubers.

Cyanide-resistant O(2) consumption can be stimulated by either treating whole white potato tubers (Norchip) with ethylene, in the presence of 100% O(2), or aging slices obtained from untreated potato tubers. A comparison of alternative pathway activity elicited by either treatment was undertaken. The proportion of electrons flowing through the alternative path in the presence of intermediate concentrations of KCN and at various concentrations of salicylhydroxamic acid was identical in both cases. However, the respiration of slices from ethylene-treated tubers was in every case stimulated by KCN, whereas the aged slices never exhibited this phenomenon. Furthermore, the metabolism of d-[U-(14)C]glucose was several hundred times greater in aged slices than in fresh slices from C(2)H(4)-treated tubers. These results, along with the respiratory kinetics of aged slices from ethylene-treated tubers, suggest that aged slices and fresh slices from ethylene-treated tubers are biochemically dissimilar.

Freeze-thaw injury to isolated spinach protoplasts and its simulation at above freezing temperatures.

Possibilities to account for the mechanism of freeze-thaw injury to isolated protoplasts of Spinacia oleracea L. cv. Winter Bloomsdale were investigated. A freeze-thaw cycle to -3.9 C resulted in 80% lysis of the protoplasts. At -3.9 C, protoplasts are exposed to the equivalent of a 2.1 osmolal solution. Isolated protoplasts behave as ideal osmometers in the range of concentrations tested (0.35 to 2.75 osmolal), arguing against a minimum critical volume as a mechanism of injury. Average protoplast volume after a freeze-thaw cycle was not greatly different than the volume before freezing, arguing against an irreversible influx of solutes while frozen. A wide variety of sugars and sugar alcohols, none of which was freely permeant, were capable of protecting against injury which occurred when protoplasts were frozen in salt solutions. The extent of injury was also dependent upon the type of monovalent ions present, with Li = Na > K = Rb = Cs and Cl >/= Br > I, in order of decreasing protoplast survival. Osmotic conditions encountered during a freeze-thaw cycle were established at room temperature by exposing protoplasts to high salt concentrations and then diluting the osmoticum. Injury occurred only after dilution of the osmoticum and was correlated with the expansion of the plasma membrane. Injury observed in frozen-thawed protoplasts was correlated with the increase in surface area the plasma membrane should have undergone during thawing, supporting the contention that contraction of the plasma membrane during freezing and its expansion during thawing are two interacting lesions which cause protoplast lysis during a freezethaw cycle.

Inhibition of o(2) consumption resistant to cyanide and its development by N-propyl gallate and salicylhydroxamic Acid.

Kinetics of inhibition of cyanide-insensitive O(2) uptake by n-propyl gallate (PG) and salicylhydroxamic acid (SHAM) were determined in fresh slices from ethylene-treated tubers of Solanum tuberosum ;Norchip' and with mitochondria and lipoxygenase (EC 1.13.11.12) isolated from these tubers. PG and SHAM appeared to be inhibiting at identical sites in mitochondria but at disparate sites in slices. The apparent K(I) for SHAM was similar in mitochondria and slices. However, the apparent K(I) for PG in mitochondria was about 40-fold lower than the K(I) for PG inhibition of lipoxygenase activity. The amount of lipoxygenase associated with mitochondria increased when tubers were treated with ethylene. PG, but not SHAM, inhibited aging-induced development of cyanide-insensitive respiration. The latter two phenomena are in accord with the hypothesis that lipid metabolism is required for the development of the alternative pathway.

Electrophysiological studies of indecainide hydrochloride, a new antiarrhythmic agent, in canine cardiac tissues.

The intracellular electrophysiological properties of a new, orally effective antiarrhythmic agent, indecainide hydrochloride, were studied in isolated canine myocardial preparations stimulated at 1 Hz and superfused with Tyrode's solution. In Purkinje fibers, indecainide (10(-6) and 3 X 10(-6) M) decreased the maximal rate of rise of phase 0 (Vmax), conduction velocity, action potential duration APD, and effective refractory period, and shifted the membrane response curve by 5 mV in a hyperpolarizing direction. In papillary muscle, APD was unchanged, but Vmax was decreased. The effect of the drug on Vmax was rate dependent, but over physiologically relevant cycle lengths (370 to 1,000 ms), Vmax remained relatively constant. In the presence of indecainide (3 X 10(-6) M) and at a basic cycle length of 333 ms, the rate constant for block onset was 0.06 and 0.1 action potentials-1 in Purkinje fiber and papillary muscle, respectively. The recovery of Vmax from maximum steady-state block was half completed in Purkinje fibers and muscle in 52 and 49 s, respectively. No resting block was apparent in either tissue at normal resting membrane potential. Indecainide had only minimal effects on automaticity arising from normal or depolarized (barium) membrane potentials. Thus, indecainide is a potent class I local anesthetic antiarrhythmic agent that depresses Vmax and conduction in cardiac tissues. The depressant effects of indecainide are completely dependent on prior activation of the tissue, but because of its slow kinetics for recovery from sodium channel block, little additional change in Vmax occurs within physiologically relevant heart rates and prematurity intervals.

[Dimensions of the human scapula]. / Contribution a l'étude des dimensions de la scapula humaine.

The study of human scapula dimensions is carried out on 110 scapulas of european adults. The statistical analysis of the results concerning the maximum height of the scapula, as well as those of the supraspinous and infraspinous fossa, and also the medial angle, show the absence of statistically significant difference between right and left sides.

Binding and pharmacologic properties of peptides derived from human and rat angiotensin II (AII) mRNA.

We investigated the binding and pharmacologic properties of peptides encoded by complementary mRNA derived from the human and rat angiotensinogen gene (human and rat IIA, respectively). Human IIA (identical with AII in 4 amino acids) inhibited binding of [125I]AII to rat adrenal glomerulosa particles (Ki = 0.62 +/- 0.09 microM) and competitively blocked, with similar potency, the ability of three AII receptor agonists to contract rabbit aorta. Rat IIA affected neither [125I]AII binding to glomerulosa particles nor the contractile response of AII. We conclude that rat IIA does not interact with AII or its receptors and that human IIA acts as a competitive inhibitor of AII at the receptor level.

Characterization of distinct angiotensin II binding sites in rat adrenal gland and bovine cerebellum using selective nonpeptide antagonists.

We investigated the characteristics of 125I-AII binding to rat adrenal and bovine cerebellar membranes in the presence and absence of new nonpeptide angiotensin II (AII) receptor ligands. The imidazole AII ligands, DUP753 and WL19, both produced biphasic competition curves to 125I-AII binding in rat adrenal glomerulosa and adrenal medulla particles, suggesting the existence of two distinct AII binding sites. Antagonist affinity (Ki) and binding capacity (Bmax) for each binding site was determined using nonlinear analysis of competition data fit to a two-site model. The high capacity site (68% of total specific 125I-AII bound) in glomerulosa had high affinity for DUP753 (4.6 +/- 0.8 nM) and low affinity for WL19 (29 +/- 3 microM), and the low capacity site had high affinity for WL19 (3.3 +/- 1.4 nM) and low affinity for DUP753 (51 +/- 9 microM). Conversely, in medulla, the high capacity site (77% total binding) had high affinity for WL19 (19 +/- 6 nM) and low affinity for DUP753 (29 +/- 8 microM), and the low capacity site had low affinity for WL19 (25 +/- 7 microM) but a high affinity for DUP753 (2.8 +/- 2.0 nM). In glomerulosa, binding parameters for the nonpeptide ligands at each site derived from monophasic competition curves obtained in the presence of either 0.3 microM DUP753 or WL19 to selectively block the high or low capacity binding site, respectively, were similar to values determined from the biphasic competition curves. Unlike the nonpeptide inhibitors, unlabeled AII yielded monophasic inhibition curves.(ABSTRACT TRUNCATED AT 250 WORDS)

Chiral recognition of pinacidil and its 3-pyridyl isomer by canine cardiac and smooth muscle: antagonism by sulfonylureas.

Pinacidil, a potassium channel opener (PCO), relaxes vascular smooth muscle by increasing potassium ion membrane conductance, thereby causing membrane hyperpolarization. PCOs also act on cardiac muscle to decrease action potential duration (APD) selectively. To examine the enantiomeric selectivity of pinacidil, the stereoisomers of pinacidil (a 4-pyridylcyanoguanidine) and its 3-pyridyl isomer (LY222675) were synthesized and studied in canine Purkinje fibers and cephalic veins. The (-)-enantiomers of both pinacidil and LY222675 were more potent in relaxing phenylephrine-contracted cephalic veins and decreasing APD than were their corresponding (+)-enantiomers. The EC50 values for (-)-pinacidil and (-)-LY222675 in relaxing cephalic veins were 0.44 and 0.09 microM, respectively. In decreasing APD, the EC50 values were 3.2 microM for (-)-pinacidil and 0.43 microM for (-)-LY222675. The eudismic ratio was greater for the 3-pyridyl isomer than for pinacidil in both cardiac (71 vs. 22) and vascular (53 vs. 17) tissues. (-)-LY222675 and (-)-pinacidil (0.1-30 microM) also increased 86Rb efflux from cephalic veins to a greater extent than did their respective optical antipodes. The antidiabetic sulfonylurea, glyburide (1-30 microM), shifted the vascular concentration-response curve of (-)-pinacidil to the right by a similar extent at each inhibitor concentration. Glipizide also antagonized the response to (-)-pinacidil, but was about 1/10 as potent with a maximal shift occurring at 10 and 30 microM. Glyburide antagonized the vascular relaxant effects of 0.3 microM (-)-LY222675 (EC50, 2.3 microM) and reversed the decrease in APD caused by 3 microM (-)-LY222675 (EC50, 1.9 microM). Nitroprusside did not alter 86Rb efflux, and vascular relaxation induced by sodium nitroprusside was unaffected by sulfonylureas. Thus, the enantiomers of the 3-pyridyl isomer of pinacidil demonstrate enhanced stereospecificity in both canine cardiac and vascular tissues compared to the enantiomers of pinacidil. However, the relative selectivity of pinacidil and its 3-pyridyl isomer for cardiac and vascular smooth muscle remains unaltered. Sulfonylureas antagonize the more potent enantiomers in both tissues, supporting the involvement of an ATP-sensitive potassium channel in the action of PCOs; however, antagonism in canine vascular smooth muscle by sulfonylureas does not resemble classical competitive antagonism.

Cellular electrophysiology of clofilium, a new antifibrillatory agent, in normal and ischemic canine Purkinje fibers.

Intracellular electrophysiological studies were performed on isolated canine cardiac tissues to investigate further the reported ability of clofilium (3 X 10(-8)--10(-6) M) to selectively increase action potential duration (APD) and refractoriness. In Purkinje fibers from normal dogs, clofilium did not influence (1) the rate of rise of the action potential (Vmax) elicited from normal or depolarized (10 mM potassium) resting potentials, (2) the Vmax of premature potentials elicited during the repolarization phase of a previous action potential or (3) the rate of diastolic depolarization of spontaneously firing Purkinje fibers. The diastolic interval was altered by inserting a single premature impulse during diastole or by varying the basic cycle length. Clofilium (3 X 10(-7) M) slightly reduced the time constant for the relation between diastolic interval and APD in concentrations that caused a maximal increase in APD of nonpremature impulses. In dogs subjected to occlusion of the left anterior descending coronary artery 48 hr before study, the APD of surviving Purkinje fibers was longer in the infarcted zone than in the normal zone. Clofilium (3 X 10(-8) M) increased APD in both zones but more so in the normal areas, thus reducing the disparity of APD between zones. Similarly, clofilium (3 X 10(-8) and 3 X 10(-7) M) increased the effective refractory period in both zones but more so in the normal area. The increase of APD and refractoriness in normal as well as depolarized or ischemic tissues in the absence of marked changes in Vmax and conduction may decrease the likelihood of reentrant arrhythmias and underlie the antifibrillatory effects in anesthetized dogs.