Protein kinase C as mediator of arachidonic acid-induced decrease of neuronal M current

The M current, I _M, of NG108-15 neuroblastoma×glioma hybrid cells, a non-inactivating K⁺ current, is decreased by arachidonic acid (5–25 M), often after an initial transitory increase. To test the possibility that the decrease is caused by activation of protein kinase C (PKC) we used the PKC 19–31 peptide, which is an effective inhibitor of PKC. With 1 M peptide in the pipette solution the normally observed strong reduction of I _M by 1 M phorbol 12,13-dibutyrate (PDB) was almost totally prevented, indicating that PKC is completely inhibited; also the voltage dependence of the M conductance, g _M(V), was shifted to more negative membrane potentials. In the presence of 1 M peptide the effect of 25 M arachidonic acid on I _M was significantly reduced, suggesting that the effect, or at least a large part of it, is mediated by PKC.     

Facilitation by 5-hydroxytryptamine of ATP-activated current in rat pheochromocytoma cells

The effects of 5-hydroxytryptamine (5-HT) on an inward current activated by extracellular ATP were investigated in rat pheochromocytoma PC12 cells. Under whole-cell voltage-clamp conditions 5-HT (10 M) reversibly enhanced the amplitude of the current activated by 30 M ATP. The enhancement may not be due to an increase in the number of functional channels because the current activated by 300 M ATP was not remarkably augmented compared with the current activated by 30 M ATP. The current enhancement by 100 M 5-HT was less obvious than that by 10 M 5-HT. When the current kinetics were compared, activation of the ATP-evoked current was accelerated to the same extent by either 10 or 100 M 5-HT, whereas deactivation was largely more accelerated by 100 M 5-HT. Propranolol (10 M), a 5-HT₁ receptor antagonist, or LY53857 (10 M), a 5-HT₂ receptor antagonist, exerted an agonistic effect: the ATP-activated current was facilitated by these compounds. Metoclopramide (10 M), a 5-HT₃ receptor antagonist, neither facilitated the ATP-activated current, nor blocked the current facilitation by 5-HT. Guanosine 5-O-(2-thiodiphosphate) (GDP[S]) (2 mM), the non-hydrolysable analog of guanosine 5-triphosphate (GTP), or K-252a (2 M), a protein kinase inhibitor, did not affect the facilitation by 5-HT of the ATP-activated current when they were included in the intracellular solution. The ATP-activated current pre-facilitated by 10 M dopamine was not enhanced by 10 M 5-HT. Similarly, the pre-facilitation by 5-HT attenuated the current enhancement by dopamine. The results suggest that 5-HT facilitates the ATP-activated channels through receptors that are not readily classified into conventional subclasses of 5-HT receptors. The reciprocal masking between the current facilitation by 5-HT and that by dopamine, combined with their sensitivities to the compounds involved in the intracellular solution, indicates that the facilitation by 5-HT may share not all, but some, common cellular mechanism with that by dopamine.     

Tetrodotoxin exerts a large frequency-dependent depression of the maximum rate of rise of action potentials in guinea pig ventricular myocytes

The action potential configuration in guinea pig ventricular myocytes was unaffected by low concentrations (0.3–1 M) of tetrodotoxin (TTX); high concentrations (10–30 M) depressed both the overshoot (5–10 mV) and duration (5–10%). Although the control was unaffected by stimulation rate (0.1–5 Hz), the depression of by TTX was greatly potentiated at rates above 1 Hz: on dose-response curves, 50% control occurred at 4.3 M (5 Hz) versus 22 M ( 1 Hz). The frequency dependent component of the depression reported here is much larger than the extra block of Na channels observed by others in voltage clamp studies on Purkinje strands. This is not a discrepancy; rather it is a consequence of a non-linear relation between and available Na conductance.     

Modulation of human neutrophil and monocyte oxidative burst byLegionella pneumophila sonic extract

The effect of Legionella pneumophila sonic extract on human neutrophil and monocyte oxidative burst was studied by Superoxide anion release and luminol-enhanced chemiluminescence assays.Legionella pneumophila sonic extract by itself did not stimulate neutrophils and monocytes. The sonic extract at 8–2000g/ml primed neutrophils for enhanced Superoxide release and, at 8–62.5 g/ml, for enhanced chemiluminescence. Monocytes were only primed for enhanced chemiluminescence at very low extract concentrations (below 16g/ml). Monocyte Superoxide release was suppressed by extract concentrations higher than 2000g/ml and the chemiluminescence response of neutrophils and monocytes by concentrations higher than 250 and 125 g/ml, respectively. The priming activity was heat stable and present in fractions below 5 kDa. On the basis of these findings it is suggested that enhanced production of oxygen metabolites by neutrophils in contact with legionella components at low concentrations could contribute to the lung tissue damage seen in Legionnaires' disease, whereas the suppression of phagocyte oxidative burst by higher extract concentrations may be one of the mechanisms by which Legionella pneumophila survives intracellularly.     

Effect of inhibitors of cyclic nucleotide phosphodiesterases on electrical and contractile activity of smooth muscle cells

Double sucrose gap experiments were carried out to study the effect of phosphodiesterase inhibitors and penetrating analogs of cyclic nucleotides on action potential and contraction of guinea pig ureteral smooth muscle cells. 3-Isobutyl-1-methylxanthine (10 M) and dibutyryl-cAMP (20 M) shortened the plateau of action potential and inhibited contraction of smooth muscle cells by increasing potassium permeability of their membrane. Vinpocetine (1 M) and dibutyryl-cAMP (100 M) strengthened contraction of smooth muscle cells and shortened action potentials by decreasing sodium permeability of their membrane.     

Comparison of disk diffusion,the E test,and detection ofmecA for determination of methicillin resistance in coagulase-negative staphylococci

The aim of this study was to find a reliable, fast, and simple alternative to the methicillin disk method for determination of methicillin resistance in coagulase-negative staphylococci, since results of this method are often difficult to read due to growth within the zone of inhibition. The sensitivity of 319 strains of coagulase-negative Staphylococci to a 5 g methicillin disk on Mueller-Hinton agar using an incubation period of 48 h was compared with that of 1 (1 g and 5 g oxacillin disks on Mueller-Hinton agar with or without 2% NaCl, using an incubation period of 24 h. The detection ofmecA (MecAgen) by the polymerase chain reaction was used as a standard. Minimum inhibitory concentrations were determined by means of the E test. Of the 225mecA-positive strains, 190, 215, and 193 were resistant to 5 g methicillin, 1 g oxacillin and 5 g oxacillin disks on Mueller-Hinton agar, respectively, and 216, 218, and 223 were resistant on Mueller-Hinton agar with 2% NaCl. Of the 94mecA-negative strains, 89, 93, and 94 were susceptible to 5 g methicillin, 1 g oxacillin, and 5 g oxacillin disks on Mueller-Hinton agar, respectively, and 92, 93, and 94 were susceptible on Mueller-Hinton agar with 2% NaCl. Using breakpoints of 2 g/ml for oxacillin resistance and 8 g/ml for methicillin resistance, the E test yielded sensitivities of 99.6 and 99.1% and specificities of 97.9 and 98.9% after 48 h of incubation. The 5 g oxacillin disk was faster and easier to read than the methicillin disk and correlated better with detection ofmecA than the methicillin disk or the 1 g oxacillin disk.     

Effects of inhibitors and ion substitutions on oscillations of cell membrane potential in cells expressing the RAS oncogene

Previous studies revealed that in NIH fibroblasts expressing the ras oncogene but not in other NIH fibroblasts, bradykinin leads to sustained, calcium dependent oscillations of cell membrane potential by repetitive activation of calcium-sensitive K⁺ channels. The present study has been performed to test for ion and inhibitor sensitivity of these oscillations. Both, Lys-bradykinin (kallidin) and bradykinin, but not any shorter peptide tested, maintained the oscillations. The oscillations are abolished in the presence of the K⁺ channel blocker barium (10 nmol/l). The amplitude but not the frequency of the oscillations is dependent on the extracellular potassium concentration. The oscillations are not dependent on the presence of extracellular sodium, bicarbonate or chloride. The oscillations are abolished in the absence of extracellular calcium and their frequency is significantly decreased at reduced extracellular calcium (to 0.2 mmol/l). The oscillations are not inhibited by acute administration of ouabain (0.1 mmol/l), by dimethylamiloride (100 mol/l), furosemide (1 mmol/l) and hydrochlorothiazide (100 mol/l), by cobalt (100 mol/l), zinc (100 mol/l), gadolinium (100 mol/l), verapamil (10 mol/l) and diltiazem (10 mol/l), but are abolished in the presence of 100 mol/l lanthanum, 1 mmol/l cadmium, 10 mol/l nifedipine, 25 mol/l SK & F 96365 and 200 mol/l TMB-8. Stimulation of calcium entry by 10 mol/l ionomycin is frequently followed by oscillations of cell membrane potential even in the absence of bradykinin. In conclusion, in cells expressing the ras oncogene bradykinin leads to sustained activation of calcium channels at the cell membrane, which cause oscillations of the cell membrane potential by triggering intracellular calcium release.     

Veratridine-induced high-frequency asynchronous release of inhibitory transmitter quanta in crayfish nerve-muscle synapses superfused with normal and low-calcium saline

Crayfish fibres of opener muscles were voltage clamped toE=–80 mV membrane potential (T=19–22°C), and veratridine (10–100 mol/l) was added to the superfusate. Within 30–60 s this caused large fluctuations of the clamp current due to vigorous asynchronous quantal release from the inhibitory nerve terminals along the muscle fibre. Excitatory postsynaptic receptors were previously desensitized by application of 5 mmol/l glutamate. Current fluctuations were evaluated by means of the noise analysis technique. Typically, 100 mol/l veratridine increased instantaneously the quantal release rate ñ from ñ<1 quantum/s toñ10,000 quanta/s. Thereafter, ñ declined exponentially with a time constant of 70s. On average, about 500,000 inhibitory quanta could be liberated in this way from the terminals on a single muscle fibre of 1 mm length. Serotonin (1 mol/l) facilitated the effect of lower veratridine concentrations (1–10 mol/l). In opener muscles veratridine-induced asynchronous quantal release showed little dependence on the bath concentration of Ca²⁺. The opposite was found for fibres of the superficial abdominal extensor muscle. Beside postsynaptic current fluctuations, veratridine elicited slowly changing average postsynaptic DC-currents which could be explained partly by superposition of individual inhibitory quantal currents. These DC-currents suggest that beside inhibitory quantal release another factor activates inhibitory postsynaptic receptors after application of veratridine.This investigation was supported by the Deutsche Forschungsgemeinschaft, SFB 220     

Phagocytic function and antibody-dependent cellular cytotoxicity of human neutrophils in the presence of N-formimidoyl thienamycin

The efficacy of an antibiotic in the treatment of bacterial infections depends upon the interactions of the drug, bacteria and phagocytes. We have studied in vitro the effect of N-formimidoyl thienamycin (Imipenem), a novel -lactamic antibiotic, on the phagocytic function and antibody-dependent cellular cytotoxicity of human neutrophil leukocytes. The incubation of these cells with 50 g/ml of Imipenem similar to the therapeutic levels reached in plasma results in an increase of their adherence capacity to nylon fiber and to substrate, induced mobility or chemotaxis, opsonization, phagocytosis ofCandida albicans (with serum, with decomplementarized serum and without serum) and latex beads, candidicidal power and the capacity of NBT reduction. Imipenem at this dose also presents chemoattractant power for neutrophils and enhances the antibody-dependent cellular cytotoxicity (ADCC).     

Activity of trimethoprim/ sulfamethoxazole plus polymyxin B against multiresistantStenotrophomonas maltophilia

The inhibitory activity of eight antibiotics and the inhibitory and bactericidal activities of combinations of trimethoprim/sulfamethoxazole (TMP/SMX) plus three fixed concentrations of polymyxin B (0.01 g/ml, 0.1 g/ml and 0.5 g/ml) against 30 multiresistant strains ofStenotrophomonas maltophilia were tested. Polymyxin B at 0.01 g/ml modified the inhibitory activity of TMP/SMX against only 40% of strains. At 0.1 g/ml and 0.5 g/ml, polymyxin B enhanced the inhibitory activity of TMP/SMX activity against all strains. Polymyxin B enhanced the bactericidal activity of TMP/SMX only at concentrations near the minimum inhibitory concentration of polymyxin B alone.     

Cytochemischer Nachweis von Katalaseaktivit?t in der Magenschleimhaut

Zusammenfassung Die Schleimhaut des Magens und des ösophagus von Mäusen wurde untersucht. Die Präparate wurden nach Fixierung in 5% igem Glutaraldehyd zum Nachweis der peroxydatischen Aktivität von Katalase in einem alkalischen (pH 9,5) DAB-Medium inkubiert.Katalaseaktivität konnte in allen Zelltypen des Epithels der Magenschleimhaut nachgewiesen werden (Mucoide Drüsenzellen, Hauptzellen, Belegzellen, Nebenzellen), sowie in den Keratinozyten des geschichteten Plattenepithels in Magen und Ösophagus. Die Enzymaktivität ist in der Matrix membranbegrenzter Zellorganellen von runder, ovaler oder stabförmiger Gestalt zu lokalisieren. (Katalase positive Partikel, CPs). Diese Partikel messen zwischen 0.1 und 0,3 m im Durchmesser, sofern ihr Anschnitt rund ist. Stabförmige Organellen können bis 1 m lang sein, wobei die Abmessungen der kurzen Achsen den Durchmessern kreisförmiger Anschnitte entspricht. Das elektronendichte Reaktionsprodukt entspricht in seinem granulären Aufbau der Struktur der mäßig elektronendichten Matrix dieser Organellen, die zu beobachten ist, wenn keine Enzymreaktion durchgeführt wurde. Die Partikel stimmen mit den in der Literatur als Mikroperoxysomen oder Peroxysomenartige Partikel beschriebenen Organellen überein.

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Cytochemical demonstration of catalase activity in the mucosa of the stomach

Summary The mucosa of the stomach and of the esophagus was investigated in mice. After fixation in 5% glutaraldehyde the tissue was incubated in an alkaline (pH 9.5) DAB-medium to demonstrate the peroxidatic activity of catalase.Catalase activity is found in all cell types of the gastric mucosa (surface epithelial cells zymogenic cells, parietal cells, mucous neck cells) and in keratinocytes of the stratified epithelium of the stomach and of the esophagus. The enzymatic activity is localized in the matrices of membrane bound organelles of round, oval or rod-like shape (catalase positive particles, CPs). Round particles measure from 0.1 m to 0.3 m in diameter. Rod-like organelles are up to 1 m long, while their short axes are similar to the mentioned diameters of round CPs. The granular and electron dense reaction product corresponds to the granular structure of a moderately dense matrix material in these organelles, which is observed in specimens which were not reacted for the demonstration of catalase activity. The catalase positive particles described in this paper resemble the microperoxisomes or peroxisome like particles described in the literature.

Diese Untersuchung wurde mit Unterstützung durch die Hochschuljubiläumsstiftung der Stadt Wien durchgeführt.     

Criteria for testing the susceptibility ofStreptococcus pneumoniae to cefotaxime and its desacetyl metabolite using 1 μg or 30 μg cefotaxime disks

In vitro susceptibility tests were performed with 350 selected strains ofStreptococcus pneumoniae to evaluate disk diffusion tests with 30 g and 1 g cefotaxime disks. Zones were compared to MICs of cefotaxime with and without its desacetyl metabolite. Cefotaxime was two to eight times more active than desacetyl cefotaxime, but the two compounds were additive when combined in vitro. For 30 g disks, zone size breakpoints were 27 mm, 28–30 mm and 31 mm for resistant, intermediate and susceptible, respectively. For 1 g disks, those zone size criteria were reduced to 13 mm, 14–16 mm and 17 mm. The 30 g disk that is currently available for testing other species can be used for testing pneumococci; however, the 1 g disk has some important advantages.     

Muscarinic modulation of calcium dependent plateau potentials in rat neostriatal neurons

Intracellular recording from neostriatal neurons in rat brain slices revealed effects of the acetylcholine (ACh) agonist carbachol (Cch, 1–10 mol/l), of the anticholinesterase physiostigmine (10 mol/l) and of the muscarinic antagonist atropine (10 mol/l) on plateau potentials elicited in the presence of K-blockers were Cadependent, elicited in the presence of K-blockers were Cadependent, since they persisted in Na-free solution, were resistant to tetrodotoxin (TTX, 3 mol/l) and blocked by Cd (0.1–0.5 mmol/l). Cch reduced the duration of the plateau potentials and made them more susceptible to fatigue. These effects were antagonized by atropine (1–10 mol/l), but not by Ba (100–200 mol/l) or 4-aminopyridine (4-AP, 0.5 mmol/l). Physostigmine (10 mol/l) had the same atropine-sensitive effects as Cch on the plateau potential. Atropine (10 mol/l), by itself, prolonged the duration of the plateau potential. High concentrations (100 mol/l) of Cch did not further reduce the duration of the plateau potential, instead, the duration re-increased with prolonged exposure. The re-increase of the plateau-spike duration was later masked by bursting activity. The opposing effects of low and high concentrations of Cch on the plateau potential duration corresponded to effects of this drug on intrastriatally evoked EPSPs in that low concentrations of Cch reduced the EPSP amplitude, but high concentrations re-increased it after a transient decrease. It is concluded that the muscarinic effect of Ach in the neostriatum is to modulate Ca-influx and that this effect is exerted in a tonic manner. On leave from absence from: Clinica Neurologica II, Universita di Roma. Tor Vergata, Roma, Italy.     

Positive cooperativity in activation of the cardiac muscarinic K+ channel by intracellular GTP

Concentration-dependent effects of intracellular GTP on activation of the muscarinic K⁺ channel were examined in inside-out patches of cardiac atrial myocytes. The pipette solution contained 0.1 M ACh. GTP (0.01–30 M) and 0.5 mM MgCl₂ were applied to the inside side of the patch membrane. K⁺ channels were activated with GTP concentration above 0.1 M. Channel activation reached a maximal value with 1–3 M GTP. It decreased at GTP concentrations larger than 3 M, probably due to desensitization. The dependence of the open probability of the channel on intracellular GTP showed a sigmoidal relationship with a Hill coefficient of around 3. A positive cooperative effect of intracellular GTP on the K⁺ channel may play an important role in amplifying the signal from the membrane receptor to the K⁺ channel.     

Inhibitory effects of E-5110 on interleukin-1 generation from human monocytes

Effects of E-5110, a novel non-steroidal antiinflammatory drug, on interleukin-1 (IL-1) generation from human monocytes were studiedin vitro. E-5110 reduced the amounts of extra- and intracellular IL-1 activity induced by lipopolysaccharide (LPS, 1 g/ml) in a dose-dependent manner (1–10M). E-5110 also inhibited the IL-1 generation induced by antigen-antibody complexes, opsonized zymosan and silica particles. It was suggested that the inhibition of IL-1 generation by E-5110 was independent of the inhibitory effects on arachidonate cyclooxygenase and/or lipoxygenase because indomethacin, piroxicam, BW755C and AA861 had no effects on IL-1 generation. Hydrocortisone (IC₅₀:0.084 M), aurothioglucose (11.5 M) and lobenzarit (75.0 M), which are clinically effective antirheumatic drugs, also inhibited IL-1 generation, like E-5110 (1.21 M). It is expected that E-5110 will be superior to classical non-steroidal antiinflammatory drugs in medical treatment of rheumatoid arthritis.     

Über das Vorkommen arterio-venöser Anastomosen im Skeletmuskel

Zusammenfassung Bei Hunden in Morphin-Urethan-Narkose wird am M. gastrocnemius mit Carnaubawachskugeln von 19, 32 und 40 mittlerer Größe das Vorhandensein arterio-venöser Anastomosen untersucht. Es ergibt sich, daß arterio-venöse Anastomosen von größerem Durchmesser als 30 äußerst selten sein dürften (meistens wurden weniger als 5% der injizierten Kugeln wiedergefunden). Dagegen wurde ein beträchtlicher Teil der injizierten 19 großen Kugeln wiedergefunden (17,5% im Mittel). Danach dürften arterio-venöse Anastomosen von mehr als 30 im Skeletmuskel selten sein und für die Durchblutung des Muskels kaum eine Rolle spielen. Es wird diskutiert, ob die zahlreichen, wiedergefundenen Kugeln arterio-venöse Anastomosen oder weite Capillaren passiert haben. Eine Entscheidung ist mit dieser Methodik nicht zu treffen.Mit 1 Textabbildung.     

Enhancement of activity of an epileptogenic focus in the frog hippocampus by kynurenins and its depression by serotonin

Experiments on frogs with an epileptogenic focus produced by injection of 1000 units penicillin (0.4 l) into the primordial hippocampus showed that preliminary injection of two kynurenins — quinolinic acid (QA, 0.1 g) and kynurenin itself (K, 1 g) — into the region of the focus or their injection into an already functioning epileptogenic focus led to an increase in the frequency of interictal epileptiform discharges and of electrographic correlates of fits on the EEG. Anthranilic acid (AA, 5 g) had no effect on activity of the focus whereas serotonin (S, 1 g) and 5-methoxytryptamine (1 g) substantially depressed it. The provoking action of the kynurenins on epileptically predisposed brain neurons, it is suggested, may play an important role in the pathogenesis of epilepsy.Department of Pharmacology, Leningrad Pediatric Medical Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR V. V. Zakusov). Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 87, No. 2, pp. 158–160, February, 1979.     

Inhibitory effects ofγ-interferon on bradykinin-induced bone resorption and prostaglandin formation in cultured mouse calvarial bones

The effects of mouse recombinant-interferon (-IFN) and indomethacin on bone resorption stimulated by bradykinin, Lys-bradykinin, Met-Lys-bradykinin, des-Arg⁹-bradykinin and prostaglandin E₂ (PGE₂) have been studied using cultures of neonatal calvarial bones and analyzing the release of⁴⁵Ca from prelabelled bones as a paramenter of bone resorption. In addition, the effects of-IFN and indomethacin on formation of PGE₂ in bone cultures stimulated by bradykinin was analyzed. Indomethacin (1 mol/l) totally abolished bradykinin (1 mol/l) induced⁴⁵Ca release. The inhibitory effect of indomethacin could be fully reversed by addition of PGE₂ (1 mol/l).-IFN (1000 U/ml) almost totally inhibited⁴⁵Ca release stimulated by bradykinin (1 mol/l), but the inhibitory effect could only be partially overcome by PGE₂.-IFN and indomethacin also inhibited the stimulatory effects of Lys-bradykinin, Met-Lys-bradykinin and des-Arg⁹-bradykinin (1 mol/l) on⁴⁵Ca release. The stimulatory effects of PGE₂ (1 mol/l) on radioactive calcium mobilization was partially inhibited by-IFN (1000 U/ml), whereas indomethacin (1 mol/l) was without effect. The inhibitory effect of-IFN on⁴⁵Ca release stimulated by bradykinin and PGE₂ was dose-dependent with threshold for action at 3–30 U/ml. Comparative dose-response curves showed that-IFN was most potent as inhibitor of bradykinin induced⁴⁵Ca release. Bradykinin (1 mol/l) significantly stimulated PGE₂ formation by a mechanism that was completely inhibited by indomethacin (1 mol/l).-IFN (1000 U/ml) partially inhibited the stimulatory effect of bradykinin on PGE₂ formation. These data show that i)-IFN is a potent inhibitor of bone resorption induced by bradykinin and bradykinin analogues and ii) that the mechanism of action can be mainly explained by an inhibition of kinin induced prostaglandin biosynthesis. The results, however, also show that-IFN can inhibit bone resorption by mechanisms unrelated to prostaglandin formation.     

Kinetics of histamine and tele-methylhistamine elimination from rat plasma

Plasma levels of histamine (Hi) and tele-methylhistamine (MeHi) were measured by HPLC after intravenous bolus injection of Hi (45 g/kg) or MeHi (55 g/kg) in the rat. Increases in plasma Hi (from baseline 2.8±0.4 to about 50 ng/ml at 2 min after the injection) were accompanied by a significant increase in MeHi levels (baseline 1.2±0.3 ng/ml rising up to 11 ng/ml, 6 min after Hi application). After rapid intravenous injection, Hi and MeHi elimination from plasma could be fitted by a double exponential function with half lives in the and phases of about 20 sec and 4 min (Hi) or 1 min and 43 min (MeHi). The results suggest that measurement of plasma MeHi, as an indirect marker of Hi release, has an advantage over measuring Hi itself.     

2 μm plasmid copy number in different yeast strains and repartition of endogenous and 2 μm chimeric plasmids in transformed strains

By using the renaturation kinetics technique we tried to get informations about the maintenance of the 2 m plasmid in yeast cells. For this purpose we determined the 2 m plasmid copy number: in various yeast strains, in a special set of mutants, in cells treated with ethidium bromide and cycloheximide and in different yeast strains obtained by transformation with 2 m chimeric plasmids.According to the strain used the proportion of 2m DNA varied from 1.1% to 3.9%, which corresponds to 24 to 88 2 m molecules per haploid genome. The particular multiresistant mutant, where the frequent loss of oligomycine resistance is correlated with the loss of extractible covalently closed circular DNA, contained 39 2 m copies per haploid genome. In the partial revertant oligomycine sensitive all the 2 m DNA sequences were lost. (Less than 0.1 copy per haploid genome.)Ethidium bromide did not affect the 2 m copy number while cycloheximide induces an increase of 36%.When a strain containing 88 2 m DNA copies per haploid genome is transformed with 2 m chimeric plasmids there is no significative change in the total number of plasmid: 36 copies of endogenous and 44 of chimeric plasmid per haploid genome. When 2 m chimeric plasmids were introduced in our 2 m-less strain despite the stability of the transformants, there is only 8 copies per haploid genome.