Spontaneously Released Research Articles

The kinetics of adenovirus infection and spread in cell cultures infected with low multiplicity.

The cause for the long "incubation period" required till the appearance of the CPE in cell cultures inoculated with small amounts of adenovirus was investigated with adenovirus type 5 in HeLa cell cultures. The spread of virus in a culture from cell to cell is minimal, as shown in cultures with antiserum in the medium. The spread by spontaneously released virus via the medium is more important. It can be accelerated by repeated subcultivation after freezing and thawing the cells in 2 or 3 days' intervals. The quantity of virus produced by one infected HeLa cell was found to be 200 TCID50 within 48 hours, independent of the MOI. The growth cycle too is largely independent of the virus dose. The data suggest that the long duration of the "incubation period" is fully explained by a burst size of 200, a cycle length of 40 to 48 hours and the assumption of a slow and steadily working process of spontaneous release of virus into the medium. Some other possible causes, like impurities in the inoculum or slow and asynchronous early stages of infection, have been ruled out by appropriate experiments.

Spontaneous release of T cell receptors for alloantigens. III. The effects of anti-T cell receptor antiserum, of alloantiserum and of trypsin on T cell receptor release.

Spleen and lymph node T lymphocytes cultivated in vitro spontaneously released receptors for alloantigens. Among the various specificities, those receptors fitting certain alloantigens could be absorbed by formolized cells or proper genotype without influencing receptors for other alloantigens. Shedding of T cell receptors could be inhibited for 8 h by treating cells with antisera directed against T cell receptors (anti-RS antisera). Inhibition experiments indicated that resynthesis of T cell receptors appears to be complete by 8 h. Experiments on prevention of receptor shedding revealed highly specific and presumably cytotoxic elimination of T cells with receptors for given alloantigens. Treatment of cells with alloantiserum obtained by skin graft rejection failed to inhibit spontaneous release of T cell receptors, whereas treatment of lymphocytes with trypsin resulted in an 8-h delay of receptor shedding similar to that after treatment of cells with anti-RS antisera. This inhibition was, however, nonspecific.

Demonstration of a genetic factor influencing spontaneous release of a xenotropic virus of mouse cells.

There is increasing evidence that mouse cells of different strains contain different numbers and biologic classes of naturally-integrated type-C RNA viruses. The present studies have examined the cellular regulation of one class of virus, xenotropic in host range, which is common to mice of NIH Swiss and NZB strains. This virus is spontaneously released at high level by NZB embryo cells but its production by NIH Swiss embryo cells is below detectable levels. Quantitative analysis of the chronic level of virus release by embryo cell lines established in tissue culture from genetic crosses of NIH and NZB strains has demonstrated the existence of a partially dominant gene influencing virus release. Evidence is presented indicating that this gene may act to provide a factor necessary for expression of a specific class of endogenous virus.

Estimation of chronic dopamine release from the caudate nucleus of the Macaca mulatta

Using a cup permanently implanted on the antero-lateral surface of the caudate nucleus, attempts were made to study the spontaneous and d-amphetamine induced release of [ 3H]dopamine (DA), which was continuously synthesised from l-[3,5- 3H]-tyrosine in the unanaesthetised Macaca mulatta placed in a restraining chair with freedom of movement of the head and members. The spontaneous release of [ 3H]DA from dopaminergic terminals was detected during the continuous superfusion of the tissue with [ 3H]amino acid in 8 monkeys. In most cases [ 3H]DA release reached a steady state level within 30 min after the onset of superfusion. Similar results were obtained when the experiments with [ 3H]tyrosine were carried out on 3 successive days. In 2 of the cases examined, the quantity of spontaneously released [ 3H]DA during the last 24 or 48 h of a 5–6 day experiment was markedly increased as compared to that observed during previous days; this effect did not appear to be related to changes in the specific activity of tyrosine in the superfusing medium. In 3 monkeys d-amphetamine ( 10 −5 M ) increased the level of [ 3HH]DA release by 15–20 times that of the normal spontaneous release of the transmitter, the effect was less pronounced in 2 other animals exhibiting a higher level of spontaneous [ 3H]DA release. The amphetamine effect could be detected every day in longitudinal studies. For example, in one animal the ratio of the amphetamine-induced [ 3H]DA release to spontaneous [ 3H]DA release varied only from 10 to 15 during a 5 day experiment. This new approach should make possible long term studies on the regulatory processes involved in DA release from dopaminergic terminals of the nigro-neostriatal system in the unanaesthetised primate.

A distinct class of inducible murine type-C viruses that replicates in the rabbit SIRC cell line.

The existence of the selectively permissive rabbit cell line SIRC allows definition of a new class of endogenous murine type-C virus. Continuous clonal lines of transformed cells derived from the BALB/c mouse-embryo cell line BALB/3T3 contain at least two distinct classes of endogenous type-C viral genomes. Spontaneously released endogenous viruses grow well on the mouse cell line NIH/3T3 (N-tropic viruses) but not on the rabbit cell line SIRC. Type-C viruses induced by treatment with BrdU grow well on SIRC (S-tropic viruses) but not in NIH/3T3 or BALB/3T3. BrdU-treated AKR mouse-embryo cells also release an S-tropic virus. N-tropic and S-tropic viruses both have the mouse intraspecies gs-1 and viral RNA-directed DNA polymerase antigenic determinants. DNA.RNA hybridization techniques reveal that the two host-range classes of endogenous viruses are only partially related to each other. Cell transformation facilitates the spontaneous release of the N-tropic viruses; treatment with thymidine analogues induces the production of the S-tropic viruses. Thus, the two classes of viral genomes appear to be subject to different cellular controls.

Spontaneous release of type C viruses from clonal lines of spontaneously transformed Blab-3T3 cells.

I wish to report that clonal lines of spontaneously transformed Balb/c 3T3 cells can spontaneously release high titres of type C virus in conditions where the untransformed cells do not release type C virus. Further, the spontaneously transformed clones of random-bred Swiss cells, such as 3T6 and 3T12 and an SV40 transformed 3T3 clone have inducible type C viruses, while in the same conditions complete virus production is not detected in the untransformed 3T3 cells. The results suggest that the control of endogenous type C virus information is altered in transformed cells.

Spontaneous release of DNA in sequential genetic order by Bacillus subtilis

The genetic activity of DNA spontaneously released during spore germination of Bacillus subtilis 168TT, was examined in a transformation system consisting of germinating spores as DNA donors and a differently marked transformable recipient. The results indicate a close correlation between DNA release and DNA synthesis. Genetic transformation using synchronously replicating germinating spores as DNA donors revealed that, in portions removed during germination, genetic markers appear extracellularly in an order similar to the known order of replication. The high relative marker frequencies obtained in transformation with released DNA allowed the construction of a genetic map often markers according to the marker frequency analysis described by Sueoka & Yoshikawa (1965) for extracted DNA. The map positions and order of replication of the various markers are in good agreement with those obtained by O'Sullivan & Sueoka (1967) and Dubnau, Goldthwaite, Smith & Marmur (1967) using density transfer experiments.

Inhibitory miniature potentials in the stretch receptor neurons of crayfish.

Intracellular microelectrodes inserted into the soma of crayfish stretch receptor neurons record frequent fluctuations of the membrane potential. Time course, amplitude, and interval distribution indicate that they are miniature potentials. At the average resting potential the polarity of the miniature potentials depends on the anion used in the microelectrode: KCl electrodes record depolarizing, K citrate or K(2)SO(4) electrodes, hyperpolarizing miniature potentials. The inhibitory postsynaptic potentials (i.p.s.p.'s) show a similar polarity change. The reversal potentials of i.p.s.p.'s and miniature potentials are equal and within 10 mv of the resting potential, more negative with K citrate (or K(2)SO(4)), less negative with KCl electrodes. Reversal can be accomplished by changing the membrane potential by stretching or by current passing. Injection of Cl(-) into the soma or replacement of external Cl by propionate results in an abrupt increase of the amplitude of the miniature potentials lasting for several minutes. The miniature potentials like the i.p.s.p.'s are reversibly abolished by the application of picrotoxin and gamma-aminobutyric acid. They are not affected by tetrodotoxin, nor by acetylocholine, eserine, or atropine. It is concluded that the miniature potentials represent a spontaneous quantal release of transmitter substance from inhibitory nerve terminals, and that the transmitter substance predominantly increases the Cl(-) permeability of the postsynaptic membrane. The effect of the spontaneously released transmitter on the behavior of the receptor neuron is considerable. The membrane conductance is increased by up to 36% and the excitability is correspondingly depressed.

Cation dependence of the noradrenaline-releasing action of tyramine

Irides were incubated for one hour in Krebs-Henseleit solution containing 10 μC/ml of dl- 3H-noradrenaline. The radioactivity spontaneously released after this period into the incubation medium consisted chiefly of acidic metabolites of noradrenaline and small amounts of normetanephrine. The increase in radioactivity during incubation with tyramine was exclusively due to noradrenaline. Omission of Ca ++ from the incubation medium caused a small but statistically significant increase both in the spontaneous and in the tyramine-induced release of radioactivity. Omission of Mg ++ had no effect. Absence of both Ca ++ and Mg ++ markedly increased the spontaneous release but reduced tyramine-induced liberation of noradrenaline. When Ca ++, Mg ++ and K + were omitted, the spontaneous release was further augmented and the effect of tyramine abolished; ouabain (10 −5M) had a very similar effect. It is concluded that in contrast to the adrenal medulla (Rubin and Jaanus, 1967) the liberation of noradrenaline by tyramine from the adrenergic nerve endings of the cat iris is not dependent on the presence of Ca ++. The releasing action of tyramine seems to be linked to a cation-dependent mechanism of tyramine transport through the membrane of the adrenergic neuron.

The release of 3H-dopamine from the isolated rabbit ileum.

1. Segments of rabbit ileum incubated with (3)H-DOPA or (3)H-dopamine and perfused with McEwen solution spontaneously release (3)H-dopamine; this basal release is doubled when the sympathetic nerves are stimulated. Evidence is presented to show that the release is probably from the sympathetic nerve terminals.2. Both cocaine and bretylium reduce the spontaneous release of (3)H-dopamine as well as the increased release resulting from sympathetic nerve stimulation. Guanethidine depresses the release during nerve stimulation but increases the spontaneous release of the amine. Phenoxybenzamine potentiates both the spontaneous release and that during nerve stimulation.3. Tyramine releases (3)H-dopamine from the preparation but tachyphylaxis to the drug rapidly develops.4. It is concluded that the release of (3)H-dopamine from the sympathetic nerves of the isolated perfused rabbit ileum possesses many of the characteristics of that of noradrenaline from other sympathetically innervated structures.

抗酸菌の溶原化に関する研究

Lysogenization rates of Mycobacteriaby mycobacteriophageswere investigated using the systems of the rapidly growing Mycobacterium, strain Jucho, and the two phages, Y 13-L and-S. Changes of bacterial characters accompanied with the lysogenization, especially phage susceptibilities and sugar availabilities, were also studied.The lysogenization rate by Y 13 S was high, 27 %, and that by Y 13 L was rather low, 4.3 %. It was shown that the phage spontaneously released from the bacterium previously lysogenized by Y 13 L, was more temperate than the original with a higher lysogenization rate of 31-47 %.The rate of spontaneous phage release of Myc. jucho (Y 13 L) was 3.5 ×10-1 and that of Myc. jucho (Y 13 S) was 1.1×10-3.The host range was studied comparatively between the orginal Y 13 L phage and its more temperate one described above, against 119 strains of rapidly growing Mycobacteria, using the RTD spot technique, and only slight differences were observed.Following changes in the phage susceptibilities of the lysogenized Mycobacteria from that of the original one were observed: Myc. jucho (Y 13 L) was resistant to the phage A 6 and D 4, and Myc. jucho (Y 13 S) was partially resistant to the phage HC, though the original strain was sensitive to those phages. Neutralization test by anti-phage sera has shown the closed relation among Y 13 L, A 6 and D 4 and between Y 13 S and HC, respectively.Acid production patterns from carbohydrates of the lysogenized mycobacteria showed no differnce from those of the original.Attempts of the phage induction of the lysogenized bacteria employing UV irradiation were carried out with no success.

THE ORIGIN OF THE ACETYLCHOLINE RELEASED SPONTANEOUSLY FROM THE GUINEA-PIG ISOLATED ILEUM.

When the guinea-pig isolated ileum had been previously treated with the anticholinesterase, NN-diisopropylphosphodiamidic fluoride (mipafox), and attached to an isotonic lever loaded with 0.5 g, it released acetylcholine into Krebs solution gassed with a mixture of 95% oxygen and 5% carbon dioxide. The amount of acetylcholine spontaneously released depended on the duration of the rest period. Cocaine, procaine or cooling the preparation to 25 degrees C greatly reduced this spontaneous output of acetylcholine, thus providing evidence in support of nervous origin of the ester. Reduction of the calcium ion content of the Krebs solution to one-twentieth of its usual value or increase in the magnesium ion content four-fold, changes which inhibit the release of acetylcholine from somatic motor nerve-endings, inhibited the output of acetylcholine from the ileum. When all the calcium of Krebs solution had been replaced by strontium one-third of the control output of acetylcholine was obtained, but the smooth muscle of the guinea-pig ileum would not respond to drugs under these conditions. Strontium could thus partially substitute for calcium in nerves but not in muscle. Hemicholinium-3 (100 mug/ml.) inhibited the spontaneous release of acetylcholine and 400 mug/ml. of choline was required to reverse the inhibition. It is concluded that cocaine, procaine, cooling, reduction of calcium ion and increase of magnesium ion concentrations all reduce the spontaneous output of acetylcholine, which has its origin in the parasympathetic nerve-endings of the intramural nerve plexuses. Further, the hemicholinium experiments seem to justify the conclusion that the release of acetylcholine is reduced because synthesis is reduced.