Preincubation of rat brain homogenates with acetylcholine (ACh) in concentrations of 10(-3)-10(-5) M for 60 minutes produces an essential increment (15-30%) in activity of microsomal Na, K-ATPase. Analogous effect was exerted by the acetylcholinesterase inhibitor eserine (10(-5)-10(-6) M). Acetylcholine has no effect in the presence of actinomycin D. Dialysis of microsomes isolated from the homogenate incubated with ACh leads to a decrease in the enzyme activity and release to the dialysate of low-molecular factor activating Na, K-ATPase of intact microsomes. The latter fact evidences the ACh-induced synthesis of activating factor and inhibition of Na, K-ATPase synthesis. After the animals are administered eserine (0.2-0.4 mg/kg), isolated microsomes show a reduced level of Na, K-ATPase (by 10-15%). Dialysis of microsomes leads to an appreciable elevation (by approximately 40%) of the enzyme activity and release into the dialysate of the inhibitory factor. The differences in the effects of eserine in vivo and in vitro suggest that during the impairment of brain integrity certain effects are excluded from the processes of the control over Na, K-ATPase activity. One of these may involve the impairment of intercellular interactions, for example, the disappearance of the effect on cholinoceptive cells of internuncial neurons that release inhibitory neurotransmitters (catecholamines).

The effect of growth conditions on urease synthesis was studied with Staphylococcus saprophyticus L-1 isolated from natural sources. Urease biosynthesis was recorded in the absence of urea in the complete medium and in the conditions of nitrogen deficiency; the highest level of the enzyme biosynthesis was found when the culture was grown in the absence of amine nitrogen in the medium. Ammonium ions were a reversible inhibitor of urease and, at a high concentration (30 g of (NH4)2SO4 per litre of the medium), partly repressed its biosynthesis. The rate of growth was low when the cells were cultivated in flasks in a medium containing urea (20 g per litre of the medium). The growth was not inhibited when the cells were cultivated in 20-litre fermenters at the same concentration of urea, but with automatic pH regulation. The alkaline medium rather than urea contained in it appeared to be the principal factor inhibiting growth of the culture.

Monocyclic and dicyclic aromatic hydrocarbons, toluene and 2-methylnaphthalene, can induce pyrocatechate-1,2-oxygenase in Candida guilliermondii BKM Y-916 cells. The enzyme is active at pH 7.0. The culture oxidizes toluene via benzoic acid by ortho-cleavage. The results indicate that Candida as an ecologo-trophic group of microorganisms can be involved, along with bacteria, in biodegradation of various aromatic hydrocarbons.

Effect of estradiol propionate on activity of seven enzymes from rabbit uterus was studied. Simultaneously with the known estrogen-induced enzymes, activity of some other enzymes from uterus cells (tyrosine transaminase, acetylcholinesterase, butyrylcholinesterase) was also studied. The hormone induced all the enzymes studied except of butyrylcholinesterase. After induction with the estrogen a new isoenzyme fraction was found in peroxidase: at the same time, content of isoenzymes of lactate dehydrogenase, tyrosine transaminase and acetylcholinesterase was increased.

Tryptophan pyrrolase (TP), which counts to the marker enzymes of the liver, failed to occur in the cells of hepatoma 22a transplanted into intact C3HA mice. In mice previously immunized with syngenic liver homogenate, 38.9% of hepatomas had a TP level reaching, on the average, 36.2% of that in the intact mice liver. Substrate induction increased the percentage of TP tumors in immunized animals up to 75%, and their average enzyme activity up to 56.9%. No hormonal induction of TP could be proved in hepatoma 22a cells.

The karyotype of mouse B-82CH-9 and B-82CH-9/CAP cell lines as well as the inheritance of their colchicine resistance in somatic cell hybrids were studied. We found earlier that these cell lines differ from other colchicine-resistant mouse and Djungarian hamster cell lines in stability of drug resistance and the lack of overproduction of the p22 polypeptide. The analysis of chromosomes stained by trypsin G-banding technique showed that, unlike mouse L-53 cells possessing unstable colchicine resistance, the B-82CH-9 and its B-82CH-9/CAP derivative had neither chromosomes with long homogeneously staining regions (HSRs) nor other cytological manifestations of gene amplification, such as double-minute chromosomes and small chromatin bodies. The hybrids of B-82CH-9/CAP and sensitive to colchicine Djungarian hamster DM-15 cells appeared in the HAT medium 10-fold more frequently than in the HAT medium containing colchicine. The hybrids grown in the HAT medium had complete chromosome complements of parent cells, while hybrids isolated from the medium with colchicine lacked some Djungarian hamster and mouse chromosomes. 4 independent clones and the cell line originated from the mixture of about 100 clones grown in HAT medium were sensitive to colchicine. We failed to transfer colchicine resistance with B-82CH-9/CAP microcells, while they were able to transfer HPRT+. At the same time, microcells of DM2/1 cells, possessing unstable colchicine resistance connected with gene amplification, could transfer both TK+ and resistance to colchicine. These results indicate that colchicine resistance of B-82CH-9 is suppressed in somatic cell hybrids. Stability of the trait, the absence of cytological manifestations of gene amplification, the lack of overproduction of p22 polypeptide and recessive inheritance suggest that colchicine resistance in these cells is not connected with gene amplification but rather results from another genetic alteration, possibly, gene mutation.

Small chromatin bodies (SCB) were revealed in Djungarian hamster cells resistant to colchicine. They looked like single bodies or like clusters of small particles. SCB were localized both in nucleus and cytoplasm. Similar formations were earlier observed in oocytes of insects with amplified extrachromosomal rDNA genes. DNA in the SCB was able to replicate not only during the S phase but also during other phases of the cell cycle. The restriction analysis showed that in cells with SCB DNA amplified sequences were replicated autonomously too. These data indicate that SCB in colchicine-resistant cells contain amplified genes. Besides, SCB double-minute chromosomes (DMs) were observed in some resistant sublines. In one of them, DMs were the only karyotypic alteration. The relationship between SCB, chromosomal homogeneously staining regions (HSRs) and DMs was studied. Single SCB and DMs appeared at the early stage of the development of colchicine-resistance (the level of drug resistance is 16-22). Selection of variants 170-220-fold resistant to colchicine was usually accompanied by the decrease in the cell number with SCB and DMs and by the increase in the amount of cells containing the chromosomes with HSRs. During the further enhancement of drug resistance (700-750), some decrease in the number of cells with HSRs and the appearance of the great number of cells containing large groups of SCB were found. The loss of colchicine-resistance observed during cultivation in colchicine free medium was accompanied by the disappearance of HSRs, emergence of SCB and DMs and further elimination of SCB and DMs from cells. The quantity of autonomously replicating amplified DNA fragments after digestive by HindIII was increased with the enhancement of SCB number in cultures.

The EcoRI and HindII restriction endonucleases and pBR325 vector plasmid were used to obtain a set of hybrid plasmids containing ColIb-P9 fragments carrying the characters for colicin Ib synthesis and immunity and the ability to inhibit T5 phage growth. The genes responsible for colicin synthesis and immunity are closely linked and localized in the EcoRI fragment with a molecular weight of 1.85 MD (pIV41) or in the HindII fragment of 2.4 MD (pIV1). The clones containing these plasmids show an increased level of both spontaneous and mitomycin C-induced colicin synthesis and an increased level of immunity due to a larger dosage of the genes. The genes controlling T5 growth inhibition are localized in other restriction fragments of ColIb DNA: the EcoRI fragment of 1.45 MD (pIV7) and the HindII fragment of 4.3 MD (pIV5). We have demonstrated by means of hybrid plasmids that T5 growth inhibition is not connected with the colicin Ib synthesized in infected cells and is controlled by other specific product(s) of the ColIb plasmid genes. T5 phage growth was as efficient in clones containing plasmids with cloned colicin Ib genes as in a strain without plasmids. An investigation of the expression of the genes inhibiting T5 phage growth in an in vitro protein synthesis system has revealed a protein with a molecular weight of 36 000 which seems to take part in the process.

The effect of various sources of carbon and nitrogen on the biosynthesis of ristomycin, protease and pigments by Nocardia fructiferi was studied. It was shown that the carbon sources had the most significant effect on the biosynthesis of the antibiotic. The maximum biosynthetic activity of the Nocardia was observed in the medium containing 1-2 per cent of soybean meal and 2 per cent of glycerol. Under such conditions all the three biologically active substances formed. The contents of ristomycin, protease and pigments amounted to 562-649 microgram/ml, 26-30 PU/ml and 0.45-0.63 conditional units, respectively.

10 independent clones of Djungarian hamster DM-15 and DMCAP cells 16-22-fold resistant to colchicine were obtained. The drug resistance was unstable. During cultivation in a colchicine free medium, 1-2% cells per population doubling lost resistance to selective dosage of the drug (0,1 microgram/ml). The loss of colchicine resistance was stepwise: the cells lost resistance to some concentrations of the drug but retained resistance to the lower colchicine dosages toxic for the wild-type cells. The analysis of chromosomes stained by the trypsin G-banding technique showed the specific karyotypic alteration in all the cells of 10 clones, i.e. complete or partial trisomy of chromosome 4. In addition, in some cells of 7 clones small chromatin bodies (SCB) and chromosomes bearing long homogeneously staining regions (HSRs) were found. The loss of colchicine resistance was accompanied by the disappearance of cells containing SCB and HSRs and the decrease in the number of cells with trisomy 4. However, the direct correlation between the loss of drug resistance and that of additional material of chromosome 4 was not observed. Probably, the resistance to colchicine is connected in these clones with gene amplification, and trisomy 4 is involved in the development of gene amplification.

The influence of ethyl methane sulfonate (methane sulfonic acid ethyl esther, EMS) on anchorage independence of tumor cells was studied. Mouse near-diploid spontaneously transformed clonal fibroblasts (CAK-25Agr were used. They were characterized by a stable low cloning efficiency in 1,2% methyl cellulose ((3-5) . 10(-5) per cell seeded into a semi-solid medium). EMS enhanced the quantity of CAK-25Agr colonies grown in methyl cellulose. However, this enhancement was only obtained when correction on the cloning efficiency of the cells in a liquid medium was introduced. Subclones of CAK-25Agr isolated from methyl cellulose were studied for their ability to form colonies in the semi-solid medium. The number of subclones with elevated anchorage independence in cultures treated by a mutagen and in untreated cultures did not differ.

The series of sublines 170-750 times more resistant to colchicine were obtained from 10 independent clones of Djungarian hamster cells possessing 16-22-fold resistance to the drug. From each clone, several sublines with different levels of colchicine-resistance were developed. The drug resistance was unstable. 2,7-4,0% of cells per population doubling lost resistance to selective dosages of colchicine. The loss of resistance was stepwise. The chromosomes stained by trypsin G-banding technique were studied in 17 sublines. 15 sublines derived from 9 independent clones contained chromosomes with long homogeneously staining regions (HSRs). These were, as a rule, primarily localized in the long arm of chromosome 4. During cultivation, HSRs were transferred from chromosome 4 into other chromosomes. Evidently, transposition of HSRs was due to translocations of different chromosomes of HSRs in the chromosome 4 and to subsequent breakages of the resulting dicentrics within HSRs. A great number of different chromosomal rearrangements was also found in the cells containing HSRs. Possibly, formation of HSR leads to destabilization of the karyotype and to the variability of the genome. The length of HSRs varied in different cells of each subline. The levels of colchicine-resistance in different sublines did not correlate with the average length of HSRs in their cells. The lack of connection between the lengths of HSRs and the levels of drug resistance as well as the existence of highly resistant sublines with gene amplification, but without HSRs, suggest that amplified genes are localized in Djungarian hamster colchicine-resistant cells both in chromosomes and extrachromosomally.

Induction of tyrosine aminotransferase using hydrocortisone was studied after administration of ethanol and CCl4 into animals. Administration of ethanol activated the enzyme. Simultaneous treatment of the animals with ethanol and hydrocortisone decreased 2-fold an inducing effect of the hormone. Within the subsequent periods the tyrosine aminotransferase activity continued to increase, exceeding the initial enzymatic activity induced by hydrocortisone. CCl4 did not affect the induction of the enzyme by hydrocortisone. The treatment with CCl4 showed an inducing effect with an increase in the enzymatic activity by 40% in liver tissue as compared with the initial level.