Joint use of light, X-ray and neutron scattering for investigation of RNA and protein mutual distribution within the 50S subparticle of E. coli ribosomes.

The values of the RNA and protein radius of gyration obtained in these studies corroborate the conclusion reported earlier [1] that on average the RNA is nearer to the center of the particle than is the protein. (It should be noted for comparison that the minimal Rg value of the RNA corresponding to its dense packing as a sphere in the center of the 52S subparticle is 49 A.) Moreover, such a great difference in the radii of gyration of RNA and protein implies a definite scheme of mutual RNA and protein arrangement in the 50S subparticle -- namely the distribution of the greater mass of proteins on the RNA surface.

A68930 is a potent, full agonist at dopamine1 (D1) receptors in renal epithelial LLC-PK1 cells.

The selective dopamine1 (D1) receptor agonists SK&F 82526 (fenoldopam) and A68930 and the mixed D1/D2 agonist SK&F 85174 were tested for their ability to stimulate adenosine 3':5'-cyclic monophosphate (cyclic AMP) accumulation in the porcine renal epithelial cell line, LLC-PK1. SK&F 82526 and SK&F 85174 were potent stimulators of cyclic AMP accumulation (EC50s 21.4 and 14.5 nM, respectively), but only partial agonists (intrinsic activities 31% and 46% of dopamine respectively). In contrast, A68930 was a potent, full agonist (EC50 12.7 nM, intrinsic activity 102% of dopamine). The stimulatory effects of A68930 and dopamine on cyclic AMP accumulation were not additive, and the stimulation of cyclic AMP accumulation by A68930 was blocked by the D1-selective antagonist, SCH 23390. These properties of A68930 suggest that it may be a useful D1-selective agonist to study renal D1 receptor mechanisms in vitro and in vivo.

Cloning of the dopamine-1A (D1A) receptor gene expressed in porcine renal epithelial cells.

We sought to determine the molecular identify of the dopamine-1 (D1) receptor expressed in the porcine renal epithelial cell line LLC-PK1. We first isolated a partial cDNA by the reverse transcription-polymerase chain reaction procedure and then used the partial cDNA to isolate positive overlapping clones from a porcine genomic DNA library. Sequence analysis of the gene revealed that the longest open-reading frame encoded a 446 amino acid protein that was 95% identical to the human D1A receptor. Expression studies in mammalian cells were also consistent with the clones encoding a D1 receptor. Northern blot hybridizations with LLC-PK1 poly (A+) RNA were strongly positive. The porcine D1A gene has two exons and a short intron in the 5' untranslated region. The 5' flanking region lacks a TATA and CAAT box but is high in GC content (68%) and contains multiple Sp1 binding sites. The 5' flanking region also contains numerous other cis-acting elements for transcription factors. These results indicate that the D1A receptor is the major D1 receptor expressed in LLC-PK1 cells and further suggest that LLC-PK1 cells may be a useful model to study the regulation of renal D1A receptor gene transcription.

[Possible mechanism of the stimulation of cell proliferation by different damaging agents]. / O vozmozhnom mekhanizme stimulirovaniia proliferatsii kletok razlichnymi povrezhdaiushchimi vozdeistviiami.

A hypothesis is advanced in which an intracellular Ca2+ increase is proposed as a common mechanism for induction of cell proliferation by different injuring influences.

[Different sensitivities of two mechanisms of excitation waves in heart tissue to anti-arrhythmia agents--blockers of fast sodium currents]. / Raznaia chuvstvitel'nost' dvukh mekhanizmov tsirkuliatsii volny vozbuzhdeniia v serdechnoi tkani k antiaritmikam--blokatoram bystrogo natrievogo toka.

In experiments with rabbit ventricular tissue sensitivity of two kinds of spiral wave sources of excitation to fast sodium current inhibition was compared. These spiral wave sources were circulation in a ring around an obstacle and circulation in tissue without an obstacle (reverberator). It was observed that after application of antiarrhythmic drugs lidocaine or mexiletine there was a prominent growth of cycle length of reverberator during first few seconds of circulation and a little change of cycle length for the circus movement around obstacle. It is proposed that different sensitivity of both kinds of spiral wave sources to antiarrhythmic drugs can be used for their distinguishing in clinical practice.

[Refractoriness of cardiac tissue and mechanism of destruction of sources of spiral waves of excitation in the heart]. / Refrakternost' serdechnoi tkani i mekhanizm gibeli istochnikov spiral'nykh voln vozbuzhdeniia v serdtse.

Ectopic spiral wave sources were produced in pieces of 15 X 15 mm of rabbit atrium by an induction of a single properly timed premature impulse. The cycle length of the ectopic source (T) gradually increased immediately after its appearance. It was shown that T growth is determined by the rise of refractoriness (R). It was found that besides the well known phenomenon of R shortening after f increase there is an opposite process of R lengthening at high driving rates (f > 4--5 sec--1). A hypothesis is advanced that the growth of refractoriness found is one of the death mechanisms of spiral wave ectopic sources in the heart.

[Mechanisms of differences of the cycle length of excitation spiral waves in atrial and ventricular tissues]. / Issledovanie mekhanizma razlichnogo povedeniia perioda spiral'nykh voln vozbuzhdeniia v predserdii i zheludochke.

Ectopic spiral wave sources--reverberators--were produced in pieces of rabbit atrium and ventricle by an induction of a single properly timed premature impulse. It was found that the cycle length (T) of the ectopic source gradually increased immediately after its appearance in atrial and changed a little bit after its appearance in ventricular tissue. Relationship between changes of T and refractorines of the heart tissues was investigated. The model is proposed which explains these changes of T by the cycle length dependence on the value of fast inward sodium current.

[Possible mechanism of long-term anti-arrhythmia and local anesthetic effect of quaternary ammonium compounds]. / O vozmozhnom mekhanizme dlitel'nogo antiaritmicheskogo i mestnoanesteziruiushchego deistviia chetvertichnykh ammonievykh soedinenii.

In experiments with dialized neurons of L. stagnalis mollusc the recovery of Na-current (INa) after its depression by local anesthetic trimecaine and its quaternary derivative N-ethyltrimecaine (G-88) was studied. A full recovery of INa within tens of seconds after washing off trimecaine but not G-88 was observed. The half-time for vanishing of INa use-dependent depression by G-88 was 17 minutes, and there was no substantial vanishing of tonic INa block even after an hour of G-88 washing off. A hypothesis is advanced that the long recovery time of INa is one of the mechanisms providing long pharmacological action of quaternary antiarrhythmic and local anesthetic ammonium compounds.

[Conformational analysis of anti-arrhythmia agents of the lidocaine series and a model for the mechanism of their action]. / Konformatsionnyi analiz antiaritmikov lidokainovogo riada i model' mekhanizma ikh deistviia.

After conformation analysis of a number of lidocaine-like antiarrhythmic molecules a simple model of their mode of action is suggested. It is proposed that the hydrophobic antiarrhythmic molecules after their absorption on cytoplasmic membranes concentrate due to polar interactions close to ionic channel proteins, then bind to these proteins and allosterically influence the process of ionic conductance.

[Refractory properties of cardiac tissue at fast sodium current decrease. Comparison of atrium and ventricle]. / Refrakternost' serdechnoi tkani pri umen'shenii bystrogo vkhodiashchego Na-toka. Sravnenie predserdiia i zheludochka.

Investigation of the membrane potential of Neurospora crassa mycelial cells was carried out by standard microelectrode technique. The resting potential is equal to--156 +/- 11 mV (negative inside the cell). The light of the spectrum blue-violet zone causes transient hyperpolarization of the cell membrane reaching --38 +/- 5 mV after 25 minutes of illumination.

[Conformation analysis and structure-activity correlations of various lidocaine-like anti-arrhythmia agents]. / Konformatsionnyi analiz i korreliatsionnoe sootnoshenie struktura-aktivnost' nekotorykh potentsial'no aktivnykh antiaritmikov lidokainovogo riada.

Structure-activity correlationship of some potential-activity antiarrhythmic agents of lidocaine-like substances is found. Conformational properties of molecules of the substances are studied.

[Effect of trimecaine, ajmaline, stenopril and chloracizine on fluctuations in K+ currents in rat liver mitochondria]. / Deistvie trimekaina, aimalina, stenoprila i khloratsizina na kolebaniia potokov K+ v mitokhondriiakh pecheni krys.

Antianginal hypotensive preparations chloracyzin and stenopryl, antiarrhythmic aimalin as well as local anesthetic and antiarrhythmic trimekaine hydrochloride were studied for their effect on the K+ inflow and outflow rate in the rat liver mitochondria during Sr2+-induced vibrations. In spite of differences in the chemical structure and pharmacological effect, all these substances are shown to uniformly suppress ion flow vibrations in mitochondria, inhibiting K+ outflow. It is found that the inhibition of the K+ outflow rate depends on the concentration of the preparations. Activity of the studied substances as to inhibition of K+ outflow from mitochondria correlates with their pharmacologic activity.

Fenoldopam is a partial agonist at dopamine-1 (DA1) receptors in LLC-PK1 cells.

Fenoldopam [6-chloro-7,8-dihydroxy-1-(4'-hydroxyphenyl)-2, 3,4,5-tetrahydro-(1H)-3-benzazepine] is a selective dopamine-1 (DA1) agonist with natriuretic/diuretic properties. A component of the natriuretic response to fenoldopam may involve direct DA1 receptor-mediated effects on proximal tubule sodium reabsorption, possibly through stimulation of adenylyl cyclase. Here, we compared the effects of fenoldopam and DA in stimulating cyclic AMP (cAMP) synthesis in LLC-PK1 cells, a renal epithelial cell line that has proximal tubule-like properties and expresses a DA1 receptor linked to stimulation of adenylyl cyclase. Fenoldopam stimulated cAMP accumulation in LLC-PK1 cells in a dose-dependent manner, an effect which could be blocked by the DA1-selective antagonist Sch 23390. Although fenoldopam was more potent than DA (EC50 55.5 +/- 7.75 nM vs. 1.65 +/- 0.64 microM) in stimulating cAMP accumulation in LLC-PK1 cells, the maximum stimulation obtained by fenoldopam was only 37% of the maximum stimulation obtained by DA(Emax 13.0 +/- 2.95 pmol/mg of protein vs. 35.6 +/- 10.19 pmol/mg of protein). Simultaneous incubation of DA and fenoldopam resulted in lower cAMP levels than with DA alone. Incubation of DA with increasing concentrations of fenoldopam produced parallel rightward shifts in the DA dose-response curves. Schild analysis further indicated that fenoldopam acted as a competitive antagonist in the presence of DA, with a pA2 value of 7.38 and a slope of unity. These results indicate that fenoldopam is a partial agonist with low efficacy at DA1 receptors linked to cAMP generation in the LLC-PK1 cells.

Locally formed dopamine stimulates cAMP accumulation in LLC-PK1 cells via a DA1 dopamine receptor.

Proximal tubules have been shown to produce dopamine (DA) from (-)-3-(3,4-dihydroxyphenyl)-L-alanine (L-dopa) and to express DA1 dopamine (DA1) receptors linked to inhibition of sodium transport. The LLC-PK1 renal epithelial cell line expresses proximal tubule cell-like properties in vitro. Here, we sought to determine whether the LLC-PK1 cell line would be a useful model system to study dopaminergic mechanisms in vitro. LLC-PK1 cells contained high levels of aromatic L-amino acid decarboxylase (AADC) (Km 0.19 +/- 0.08 mM, Vmax 3.69 +/- 0.57 nmol.mg-1.min-1) and converted L-dopa to DA in a nonsaturable fashion up to 1 mM L-dopa. DA production was blocked by the AADC inhibitor carbidopa. Dopamine stimulated adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in LLC-PK1 cells in a dose-dependent manner (50% effective concentration, 1.53 +/- 0.38 microM; maximal stimulation, 46.6 +/- 10.88 pmol/mg protein); this effect was blocked by addition of DA1-receptor antagonists. L-Dopa also stimulated cAMP accumulation, and this effect was attenuated by an equimolar concentration of carbidopa and blocked by the DA1 antagonist Sch 23390. These results indicate that LLC-PK1 cells convert L-dopa to DA, which then stimulates cAMP via a DA1 receptor coupled to activation of adenylate cyclase. Moreover, the demonstration that locally formed DA can act as an autocrine/paracrine substance in LLC-PK1 cells in vitro is consistent with a role for DA as an autocrine/paracrine substance in vivo.

On the distribution and packing of RNA and protein ribosomes.

From the analysis of the measured radii of gyration of the RNA (Rg = 6.6 +/- 0.3 nm) and protein (Rg = 10.2 +/- 0.5 nm) components of the 50-S subparticle of Escherichia coli ribosomes it is concluded that proteins containing a large amount of hydrodynamically bound water are located on the periphery of the tightly packed RNA. We found that the common features of the measured X-ray scattering curves of the E. coli 70-S ribosome, its 30-S and 50-S subparticles and wheat 80-S ribosomes in the region of scattering angles corresponding to scattering vectors mu from 1 to 5 nm-1 reflect features of the RNA compact packing. A hypothesis is proposed that the compact packing of RNA helices in the range of Bragg distances of 4.5--2.0 nm is a general structural feature of all ribosomal particles.

A vancomycin-inducible lacZ reporter system in Bacillus subtilis: induction by antibiotics that inhibit cell wall synthesis and by lysozyme.

We have constructed a Bacillus subtilis strain in which expression of a vanH::lacZ gene fusion is regulated by VanR and VanS of Enterococcus faecium. This construct allows a nonpathogenic bacterial strain to be used as a model system for studying regulation of vancomycin resistance. Antibiotics and enzymes that affect cell wall biosynthesis and stability were tested for the ability to induce lacZ expression. As a result, fosfomycin and D-cycloserine were added to the group of peptidoglycan synthesis inhibitors shown to induce expression from the vanH promoter. Induction by cell wall hydrolytic enzymes, as well as by antibiotics whose actions may lead to the accumulation of chemically different peptidoglycan precursors, raises the possibility that models that postulate induction by peptidoglycan [correction of peptidodoglycan] precursors are wrong.

Prostaglandin E2 production in rat IMCD cells. II. Possible role for locally formed dopamine.

Dopamine has been proposed as an intrarenal natriuretic hormone. We reported previously that inner medullary collecting duct (IMCD) cells express a novel DA2-like dopamine receptor (namely, DA2K) that is linked to stimulation of prostaglandin E2 (PGE2) production. In this study we examined whether locally formed dopamine could stimulate PGE2 production in cultured IMCD cells. L-Dopa stimulated PGE2 production dose dependently in cultured IMCD cells (concentration for half-maximal stimulation, 54.3 microM; maximal stimulation, 212.7% of basal), with the maximal stimulation similar to that obtained with dopamine. This effect was blocked by aromatic L-amino acid decarboxylase (AADC) inhibitors and DA2-receptor antagonists. IMCD cells also had measurable AADC activity and produced dopamine from exogenously added L-dopa. AADC inhibitors and DA2 antagonists also lowered basal PGE2 levels, suggesting that dopamine was being formed constitutively in culture. These results suggest that cultured IMCD cells have the capacity to take up and convert L-dopa to dopamine, which then stimulates PGE2 production via DA2K receptors. These results further suggest that locally formed dopamine could act as an autocrine/paracrine hormone in the kidney inner medulla to regulate PGE2 synthesis and water and electrolyte excretion.