Hybridization of bean, spinach, maize and Euglena chloroplast transfer RNAs with homologous and heterologous chloroplast DNAs. An approach to the study of homology between chloroplast tRNAs from various species.

Chloroplast tRNAs from two dicotyledons (spinach and bean), a monocotyledon (maize) and a green alga (Euglena) have been fractionated by two-dimensional gel electrophoresis. The individual tRNAs have been identified, albeled with 125I or 32P, and used in tRNA-DNA hybridization experiments. Spinach chloroplast tRNAs hybridize as well, and maize chloroplast tRNAs almost as well as bean chloroplast tRNAs to bean chloroplast DNA, thus suggesting a high degree of homology between the chloroplast tRNAs from the two dicotyledons and between the tRNAs from the two dicotyledons and those of the monocotyledon. But Euglena total chloroplast tRNA hybridizes very poorly to bean chloroplast DNA, and among the 14 individual tRNAs tested, only one, Euglena chloroplast tRNAPhe, hybridizes to both maize and bean chloroplast DNAs, which is in good agreement with the fact that Euglena and bean chloroplast tRNAsPhe have almost identical primary structures.

Construction of the physical map of the chloroplast DNA of Phaseolus vulgaris and localization of ribosomal and transfer RNA genes.

Construction of a physical map of the chloroplast DNA from Phaseolus vulgaris showed that this circular molecule is segmentally organized into four regions. Unlike other chloroplast DNAs which have analogous organization, two single-copy regions that separate two inverted repeats have been demonstrated to exist in both relative orientations, giving rise to two populations of DNA molecules. Hybridization studies using individual rRNA and tRNA species revealed the location of a set of rRNA genes and at least seven tRNA genes in each inverted repeat region, a minimum of 17 tRNA genes in the large single-copy region and one tRNA gene in the small single-copy region. The tRNA genes code for 24 tRNA species corresponding to 16 amino acids. Comparison of this gene map with those of other chloroplast DNAs suggests that DNA sequence rearrangements, involving some tRNA genes, have occurred.

Fractionation and identification of spinach chloroplast transfer RNAs and mapping of their genes on the restriction map of chloroplast DNA.

Spinach chloroplast 4S RNAs has been separated by two-dimensional polyacrylamide gel electrophoresis into about 35 species. After extraction from the gel, 27 of these RNA species were identified by aminoacylation as tRNAs specific for 16 amino acids. Individual tRNAs were labeled in vitro with 125I and hybridized to DNA fragments obtained by digestion of spinach chloroplast DNA with KpnI, PstI, SalI and XmaI restriction endonucleases. A minimum of 21 genes corresponding to tRNAs for 14 different amino acids have been localized on the restriction endonuclease cleavage site map of the DNA molecule. Of these, 15 genes corresponding to tRNAs for 12 amino acids are located in the larger of the two single-copy regions which separate the two inverted copies of the repeat region. Each copy of this repeat region contains a set of genes for the ribosomal RNAs and a gene for tRNA2Ile in the "spacer" sequence between the 16S and 23S ribosomal RNAs. The genes for tRNA1Ile, tRNA2Leu and tRNA3Leu also map in the repeat region, but outside the ribosomal DNA unit. At present, two more chloroplast tRNAs (for Pro and Lys) have been identified, but not mapped, while 4 unidentified 4S RNAs have been mapped in the large single-copy region of the DNA molecule. Evidence is presented that isoaccepting tRNA species can be transcripts from different loci.

Age-related variations in tissue angiotensin converting enzyme activities: comparison between spontaneously hypertensive and Wistar-Kyoto rats.

Angiotensin converting enzyme (ACE) activity was measured by fluorimetry in the plasma, lung, heart, aorta and kidney (cortex and medulla) of 3-, 5-, 8- and 11-week-old spontaneously hypertensive rats (SHR) and compared with that of age-matched Wistar-Kyoto rats (WKY). In the plasma, lung and kidney (cortex and medulla), ACE activity was lower in SHR than in WKY. This was evident as early as the age of 3 weeks. In contrast, there were no differences between SHR and WKY in the aorta and the heart. Age-related variations in ACE activities differed in each tissue and in both groups of rats, but no major modifications were correlated with the development of hypertension. A binding assay was performed with [3H]ramiprilat; affinity (KD) and the maximum number of binding sites (Bmax) were determined in plasma and tissues of 3-week-old SHR and WKY. The KD values were identical in the two groups but Bmax was lower in all SHR tissues except in the heart; these results might be related to the decrease in ACE activity. Our results probably reflect genetic differences in ACE activity between SHR and WKY, and suggest that ACE regulatory mechanisms act differently in each tissue.

Ca2+ channel inhibition in a rat osteoblast-like cell line, UMR 106, by a new dihydropyridine derivative, S11568.

UMR 106 rat osteogenic sarcoma cells were studied with the whole cell patch clamp technique to investigate the presence of voltage-gated inward currents. In barium (Ba2+)-containing medium, depolarizing jumps revealed both transient (T-type) and sustained (L-type) Ba2+ currents. The L-type component was dihydropyridine-sensitive: the agonist Bay K 8644 increased the amplitude of the L-type Ba2+ current. A new dihydropyridine calcium channel blocker, S 11568 ((+/-)-2(2-[2-(aminoethoxy)ethoxyl]methyl)4-(2',3'- dichlorophenyl)3-ethoxycarbonyl-5-methoxycarbonyl-6-methyl-1,4- dihydropyridine, and its enantiomers, S 12967 ((+)-S 11568) and S 12968 ((-)-S 11568), inhibited the L-type Ba2+ current. IC50 values at a holding potential (VH) of -50 mV were 90 nM for S 11568, 800 nM for S 12967 and 45 nM for S 12968. At VH = -80 mV, S 12968 was less potent (IC50 near 500 nM). In contrast, S 12968 was without appreciable effect on the T-type component of the inward current through Ca2+ channels. Our results indicate that UMR 106 cells express both T-type and L-type Ca2+ channels and could be used to study the modulation by Ca2+ channel blocking agents, such as S 12968, of the hormonal regulation of Ca2+ fluxes across the osteoblast membrane.

Characterization of a high affinity piretanide receptor on kidney membranes.

The tritiated loop diuretic, piretanide, is a useful ligand for specific diuretic receptors which are present in the plasma membranes of renal medullary cells. Its high specific activity (30 Ci X mmol-1) made it possible to demonstrate the existence of a high affinity receptor (Kd approximately 5 nM) and a binding site with low affinity. High affinity binding is saturable, reversible and displaceable by a number of non-radioactive loop diuretics. Structural analogues, devoid of diuretic activity, do not displace piretanide binding. No specific binding occurs in liver or spleen membranes.

Renal and iliac vascular effects of dopamine in the anaesthetized rat.

The renal and iliac vascular effects of dopamine were compared in pentobarbital anaesthetized rats. Local vascular resistances were calculated from simultaneous measurement of blood pressure, renal and iliac blood flow. Without pretreatment, dopamine increased renal and iliac vascular resistance. After pretreatment with prazosin, dopamine decreased the renal vascular resistance while the iliac vascular resistance was still increased. After a combination of yohimbine and prazosin pretreatment, dopamine lowered both the renal and iliac vascular resistance by 30%. These responses were not modified by the beta-adrenoceptor antagonist, sotalol, or by pretreating the rats with reserpine. The renal but not the iliac vascular response to dopamine was abolished by (+)-butaclamol, a stereoselective dopamine receptor antagonist, and by SCH 23390, the DA1-selective dopamine receptor antagonist. The decrease in iliac vascular resistance was not modified by indomethacin or the non-selective 5-HT receptor antagonist, metitepin. These results show that after blockade of alpha 1 and alpha 2-adrenoceptors, dopamine induces iliac vasodilation by a postsynaptic mechanism independent of an interaction with beta-adrenoceptors, dopamine or serotonin receptors. They also confirm in the rat in vivo the existence of renal vasodilation mediated by DA1 dopamine receptors.

Characterisation of the alpha-adrenoceptors of the rat renal vascular bed.

Postjunctional renal alpha-adrenoceptors were studied (1) in vivo, on the renal vasculature of the anaesthesized rat and compared with those in the femoral vasculature, and (2) in vitro, on the renal vascular bed of isolated perfused rat kidney. In vivo, renal and iliac blood flows were measured with an electromagnetic flow meter. The i.v. injection of (-)-phenylephrine (1-16 micrograms/kg) and B-HT 920 (0.6-600 micrograms/kg) induced an increase in both renal and iliac vascular resistance, inhibited respectively with prazosin (300 micrograms/kg) or yohimbine (300 micrograms/kg). In the kidney, maximum response to B-HT 920 was equivalent to 64% of that to (-)-phenylephrine; on the iliac vasculature, vasoconstrictor responses to both drugs were identical, but only corresponded to 50% of the maximum renal response to (-)-phenylephrine. This indicates the predominance of alpha 1- over alpha 2-adrenoceptors in the renal vascular bed. In vitro, on the isolated perfused rat kidney, vasoconstriction was induced by the preferential alpha 1-adrenoceptor agonists [(-)-phenylephrine, cirazoline and methoxamine] and the preferential alpha 2-adrenoceptor agonists (alpha-methylnoradrenaline, dopamine and clonidine) at concentrations at which they lose their selectivity for the alpha 2-adrenoceptors; all responses were antagonised by prazosin but not by yohimbine. B-HT 920, the selective alpha 2-adrenoceptor agonist, only induced renal vasoconstriction in vitro under concomitant infusion of rabbit plasma.

The antagonist properties of S 11978 on 5HT3 receptors in N1E-115 neuroblastoma cells.

The effects of the novel antagonist S 11978 (Endo-7-[(8-methyl-8-azabicyclo[3,2,1]-3-octyl)oxycarbonyl] benzo[b] thiophene) on 5HT3 receptors were examined in N1E-115 mouse neuroblastoma x rat glioma hybrid cells, with radioligand binding and whole cell patch clamp techniques. The 5HT3 receptor ligand [3H] quipazine was displaced by ICS 205-930, GR 38032F and S 11978 with KI values of 2.25 nM, 36.5 nM and 1.75 nM respectively. Electrophysiological studies showed that S 11978 is a potent 5HT3 antagonist: IC50 values for inhibition of 5HT-induced inward current by ICS 205-930, GR 38032F and S 11978 were 0.22 nM, 0.63 nM and 0.43 nM respectively at a holding potential of -65 mV. It is concluded that S 11978 is a potent, high affinity 5HT3 receptor antagonist.

Binding of loop diuretics to their renal receptors: use as a screening model for potential diuretic activity.

Loop diuretics of the benzoic acid and aryloxyacetic acid families inhibit Na+K+Cl- cotransport. The ranking order of potencies measured in the thick ascending limb of Henle's loop and the ranking order of affinities for [3H]piretanide receptors on renal plasma membranes are the same. Potencies and affinities correlate well (correlation coefficient r = 0.959 for the medulla and r = 0.951 for the cortex). Therefore, measurement of [3H]piretanide binding is proposed to facilitate screening for loop diuretic action.

Phenotypic instability of Salmonella strain YG1024 during mutagenicity assays of arylamine promutagens.

During spot tests using Salmonella TA98 derivatives (YG1021, YG1024) and TA100 derivatives (YG1026, YG1029), a unique response of O-acetyltransferase (OAT)-enhanced strains YG1024 and YG1029 to arylamines was observed. On plates containing rat-liver S9, these strains yielded revertant colonies induced in two separate concentric rings around the site of application, while the parent (TA98, TA100) and nitroreductase-enhanced strains (YG1021, YG1026) did not exhibit this response. The inner ring of revertants was accompanied by cytotoxicity and microcolony formation, with the outer ring in a region without background lawn toxicity. Addition of tetracycline to the top agar eliminated formation of the inner ring of YG1024 revertants in spot tests and reduced the revertant count in preincubation assays at cytotoxic dose levels of 2-aminoanthracene, 2-aminofluorene, 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline. Tetracycline sensitivity indicates that mutant colonies developing at high concentration/toxicity arose, in effect, from TA98 regenerated by functional loss of the tetracycline-resistance plasmid (pYG219) from YG1024. Mutant colonies found at low concentration/toxicity arose from normal plasmid-bearing YG1024. These results indicate the need to consider coincidental toxicity-induced instability in YG1024 during quantitative mutagenicity assays of arylamines and uncharacterized complex mixtures.

[Comparison of the vasomotor effects of dopamine on the renal and iliac arterial beds of the anesthetized rat]. / Comparaison des effets vasomoteurs de la dopamine sur les lits artériels rénal et iliaque chez le rat anesthésié.

Dopamine remains the reference in the study of the mechanisms involved in the antihypertensive effects of dopaminomimetics. Its renal vasodilator effects are well characterized but relaxation of other vascular beds is less known. The iliac vascular response to dopamine was studied in the anesthetized rat (pentobarbital) and compared to the renal response. Simultaneous measurements of arterial pressure, iliac and renal blood flows (electromagnetic flowmeter probes, Skalar, Delft) allowed iliac and renal vascular resistance (IVR, RVR) to be calculated. Their variations were studied after intravenous injections of increasing doses of dopamine (1.5 to 200 micrograms/kg) in unpretreated animals and in animals receiving various pretreatments. Without pretreatment, dopamine induced a biphasic renal response, vasodilation partially masked by subsequent vasoconstriction for doses above 12.5 micrograms/kg of dopamine. Simultaneously, IVR was increased. After alpha-adrenolytic pretreatment (prazosin 2.5 mg/kg, i.v.), dopamine decreased the RVR while iliac vasoconstriction persisted. The association of yohimbine (5 mg/kg, i.v.) to prazosin completely abolished the vasoconstrictive effects: dopamine lowered then by about 30% both IVR and RVR. Dopamine-induced iliac and renal decrease in vascular resistance persisted in the presence of a beta-adrenoceptor antagonist [+/-)-sotalol 30 mg/kg, i.v.), after depletion of catecholamines from sympathetic terminals (reserpine 10 mg/kg, i.p. 20 h before the experiment) or inhibition of cyclo-oxygenase (indomethacin 2.5 mg/kg, i.p. 20 h and 1 h before dopamine). On the contrary, a specific antagonist of dopamine receptors, (+)-butaclamol (60 micrograms/kg/min, i.v.) stereoselectivity inhibited the dopamine-induced renal vasodilation but did not modify the iliac response.(ABSTRACT TRUNCATED AT 250 WORDS)

[Effects of conversion enzyme inhibitors on the synthesis of angiotensin II by the isolated rat kidney]. / Effets des inhibiteurs de l'enzyme de conversion sur la synthèse d'angiotensine II par le rein de rat isolé.

On the isolated perfused rat kidney, angiotensin-converting-enzyme activity was evaluated by two approaches: one, biochemical, through the measurements of the enzymatic activity on renal homogenate, the other, pharmacological, through the vasoconstrictor response to angiotensin I. Renal tissue angiotensin-converting-enzyme activity was not modified by setting the kidney under perfusion with a modified Krebs-Henseleit solution but was inhibited after addition of captopril into the perfusion medium (10(-5) M, 100 p. 100 inhibition) or after pretreatment of the animals with ramipril (10 mg/kg/day over 3 weeks, per os, 60 p. 100 inhibition). On the isolated perfused rat kidney, angiotensin I and angiotensin II induced a concentration dependent renal vasoconstriction (EC50 = 1.05 +/- 0.18 X 10(-8) and 0.11 +/- 0.05 X 10(-8) M) which was competitively antagonized by saralasin, an angiotensin II receptor antagonist. Addition of angiotensin-converting-enzyme inhibitors to the perfusion medium (captopril or ramiprilat, 10(-5) M) or pretreatment of the animals with ramipril (50 mg/kg, i.p. the day before or 10 mg/kg/day over 3 weeks, per os) only shifted the angiotensin I concentration-response curve to the right by a factor 3 to 4. The residual vasoconstrictor effect of angiotensin I was abolished by 10(-5) M saralasin and remains linked to a local generation of angiotensin II. Our results suggest that, on the isolated perfused rat kidney, besides the angiotensin-converting-enzyme, an iso-enzyme may also be able to generate angiotensin II.

Patterns of change in adolescent smoking behavior and results of a one year follow-up of a smoking prevention program.

A smoking prevention program for adolescents conducted in two public middle schools focused on resisting peer pressure to smoke and understanding the intent of commercial cigarette advertising. One class in each school participated in the program group and one served as a control group. The program consisted of eight sessions and was conducted by first-year medical students. Data on smoking behavior and related information were obtained from self-administered questionnaires at baseline, at the conclusion of the program, and one year later. One year after the program was concluded, the proportion of non-smokers was higher among those who had participated in the program than among the controls. This suggests that routine implementation of smoking prevention programs in conventional school settings may be productive in reducing the prevalence of cigarette smoking.

Loop diuretics bind to distinct receptors in renal medulla and cortex.

Receptors which bind [3H]-piretanide were demonstrated on membranes prepared from renal medulla and cortex. They exhibit all the characteristics of a specific receptor such as high affinity, specificity, tissue selectivity, reversible binding kinetics and proportionality between affinity and diuretic effect. Medullary and cortical receptors are clearly distinct since they have different ion requirements.