[The study of molecular mechanisms of action of natural amino acids and serotonin on adenylyl and guanylyl cyclases of the ciliates].

It has been previously shown that some amino acids and their derivatives are capable of regulating the activity of adenylyl cyclase (AC) and guanylate cyclase (GC) in free-living ciliates Dileptus anser and Tetrahymena. The aim of this work was to study the molecular mechanisms of action of methionine, tyrosine, alanine and neurohormone serotonin on the activity of enzymes-cyclases and the identification of their specific receptors in D. anser and T. pyriformis. Methionine and serotonin significantly increased the basal AC activity in both ciliates, and the AC effect of serotonin in T. pyriformis was carried out with the participation of Ca2+-dependent form of AC and heterotrimetic G proteins. AC stimulating effect of tyrosine and alanine was expressed weakly and only detected in D. anser. Serotonin is both ciliates and alanine in D. anser stimulated GC activity, whereas methionine and tyrosine had no effect on GC. Methionine and serotonin bind to surface receptors of the ciliates with high affinity. K(D) for [methyl-3H] methionine binding to D. anser and T. pyriformis were 7.5 and 35.6 nM, and for [3H] serotonin binding were 2.7 and 4.7 nM, respectively. Alanine and tyrosine bind to the ciliates with low affinity. Thus, ciliates D. anser and T. pyriformis have chemosignaling systems regulated by amino acids and their derivatives and including the enzymes with cyclase activity. There is an assumption that these systems are similar to hormonal signaling systems of higher eukaryotes and are their predecessors.

[Effect of natural amino acids and sugars on cyclase activities in infusoria Tetrahymena pyriformis and Dileptus anser].

Natural amino acids and sugars in intracellular eukaryotes are known to regulate adenylyl cyclase (AC) and guanylyl cyclase (GC) systems that control the most important cell processes. The goal of the present work consisted in study of effects of natural amino acids and sugars and some of their derivatives on AC and GC activities of infusoria Tetrahymena pyriformis and Dileptus anser. Methionine, arginine, lysine, and tryptamine stimulated basic AC activity of T. pyriformis, whereas alanine, thyrosine, and cysteine decreased it. Methionine, glycine, alanine, thyrosine, arginine, and to the lesser degree tryptamine and histidine stimulated AC of D. anser. The GC activity of T. pyriformis are increased in the presence of tryptamine, tryptophane, histidine, arginine, and lysine, whereas glycine and aspartic acid, on the contrary, decreased it. Tryptamine, tryptophan, leucine, glutamic acid, serine, histidine, and alanine stimulated the GC activity of D. anser. Glucose, fructose, and sucrose stimulated the basal AC activity of both infusorians and GC of T. pyriformis, with glucose and sucrose increasing AC of T. pyriformis twice, while that of D. anser 4.5 times. Lactose stimulated AC and GC of T. pyriformis and was inefficient with respect to the D. anser cyclases, whereas mannose and galactose did not affect the enzyme activities in both infusorians. The study of the chemotactic response of infusorians to amino acids and sugars indicates that involved in realization of this response can be signaling pathways both dependent on and independent of cyclic nucleotides. Thus, it has been established for the first time that several amino acids and sugars affect functional activity of enzymes with cyclase activity of the infusorians T. pyriformis and D. anser. This confirms the hypothesis that at early stages of evolution the large spectrum of comparatively simple natural molecules has a hormone-like action.

[Regulation by cyclic adenosine monophosphate of functional activity of the adenylyl cyclase system of the infusorian Dileptus anser].

In some unicellular eukaryotes, cAMP performs functions not only of the second messenger, but also of hormone, the primary messenger. We have found that cAMP binds to surface receptors of the free-living infusorian Dileptus anser and stimulates activity of the adenylyl cyclase signaling system (AC-system) including heterotrimeric G-proteins and enzyme adenylyl cyclase (AC). The binding of cAMP to receptor is performed with a high affinity (K(D), 27 nM) and is highly specific, as cGMP and adenosine do not produce a marked effect on it. The infusorian cAMP-receptors have been shown to be coupled to G-proteins, which is indicated by a decrease of their affinity to the ligand in the presence of GTP, stimulation of the GTP-binding of G-proteins with the cyclic nucleotide, and block of the cAMP regulatory effects with suramin, an inhibitor of heterotrimeric G-proteins. cAMP stimulates dose-dependently the AC activity, its effect remaining virtually unchanged in the presence of cGMP, AMP, GMP, and adenosine. N6,O2-dibutyryl-cAMP, a non-hydrolyzed cAMP analog, only at comparatively high concentrations competes with cAMP for binding sites and decreases the cAMP stimulating effects on the AC activity and GTP binding. Thus, we have shown for the first time that the AC system of the infusorians D. anser is stimulated with the extracellular cAMP that in this case functions as the external signal regulates activity of extracellular cAMP-dependent effector systems.

[Functional characteristics of calcium-sensitive adenylyl cyclase of ciliate Tetrahymena pyriformis].

Calcium-sensitive forms of adenylyl cyclase (AC) were revealed in most vertebrates and invertebrates and also in some unicellular organisms, in particular ciliates. We have shown for the first time that calcium cations influence the AC activity of ciliate Tetrahymena pyriformis. These cations at the concentrations of 0.2-20 microM stimulated the enzyme activity, and maximum of catalytic effect was observed at 2 microM Ca2+. Calcium cations at a concentrations of 100 microM or higher inhibited the AC activity. Calmodulin antagonists W-5 and W-7 at the concentrations of 20-100 microM inhibited the catalytic effect induced by 5 microM Ca2+ and blocked the effect at higher concentrations of Ca2+. Chloropromazine, another calmodulin antagonist, reduced Ca2+-stimulated AC activity only at the concentrations of 200-1000 microM. AC stimulating effects of serotonin, EGF and cAMP increased in the presence of 5 microM Ca2+. AC stimulating effects of EGF, cAMP and insulin decreased in the presence of 100 microM Ca2+, and AC stimulating effect of cAMP decreased also in the presence of calmodulin antagonists (1 mM). At the same time, stimulating effect of D-glucose in the presence of Ca2+ and calmodulin antagonists did not change essentially. The data obtained speak in favor of the presence of calcium-sensitive forms of AC in ciliate T. pyriformis which mediate enzyme stimulation by EGF, cAMP, insulin, and serotonin.

[Change of mating type induced by actinomycin D in the ciliate Dileptus anser].

Effect of actinomycin D on the expression and inheritance of mating types (MTs) in mature laboratory clones of the ciliate Dileptus anser was studied. Each mature clone of these ciliates isolated from natural population appears to belong to one of the three complementary MTs - MT I, MT II or MT III. Its MT does not vary in the course of further laboratory cultivation of the clone in a series of vegetative generations. However, after treatment with actinomycin D (15 microg/ml, 3 days), such clones became hereditarily unstable for their MTs. At weekly testing for MT over the course of at least 15 weeks after treatment (which corresponds to more than 100 cellular divisions), many subclones of the treated clone reversibly changed their MT for another, and (or) showed temporary state of immaturity or partial maturity. These data testify in favour of our hypothesis of epigenetic MT determination in D. anser. Considering that actinomycin D induces heritable destabilization of some characters in amebas Amoeba proteus which is obviously of epigenetic nature, this antibiotic can possibly be regarded as "epimutagen".

[Inhibition of the adenylyl cyclase signaling system by colchicine and vinblastine in ciliate Dileptus anser].

Cytoskeleton plays a key role in functioning of hormonal signaling systems in vertebrate animals. The data on the influence of cytoskeleton components, in particular the microtubules, on functional activity of chemosignaling systems of unicellular organisms are absent at the present time. The aim of this work was the study of influence of microtubule-disrupting agents, colchicine and vinblastine, on the adenylyl cyclase (AC) system of free-living ciliate Dileptus anser. The treatment of D. anser with colchicine and vinblastine (10(-5)-10(-6) M) weakly influenced basal activity of AC, but caused essential decrease or complete blocking of AC stimulation by non-hormonal (GppNHp, sodium fluoride) and hormonal (aldrenaline, serotonin, glucagon) agents. As a result of this treatment, a decrease of the basal level of GTP-binding of heterotrimeric G-proteins and inhibition of G-protein stumulation by the hormones were found. In the case of adrenaline it was that colchicine and vinblastine disturb the AC stimulation by the hormone, mediated with the G(s)-protein, but weakly influence its inhibitory AC effect, realized via the G(i)-protein. Thus, it was established for the first time that microtubules in unicellular organisms such as the ciliate D. anser are involved in regulation of functional activity of AC system, and their action is realized at the level of the G-proteins similar to the G(s)-proteins of the vertebrates.

[Regulatory calcium effect on adenylyl cyclase functional activity in the infusorian Dileptus anser].

Earlier we have shown that some non-hormonal activators of adenylyl cyclase (AC) and hormones of higher vertebrate animals are able to affect functional activity of the AC system in the infusorian Dileptus anser. In the present work, sensitivity of this infusorian AC to Ca2+ was studied and it was found that calcium cations at concentrations of 0.5-10 microM stimulated significantly the enzyme activity in D. anser partially purified membranes. An increase of Ca2+ concentrations to 100 microM and higher led to the complete block of their stimulatory effect. In the EDTA-treated membranes the enzyme activity was reduced markedly, but it was restored significantly by addition of Ca2+. Calmodulin antagonists--chlorpromazine, W-7, and W-5--caused a dose-dependent decrease of the enzyme activity stimulated by 5 microM Ca2+ with IC50 values of 35, 137, and 174 microM, respectively. The AC-stimulating effects of biogenic amines (serotonin and octopamine) were completely retained in the presence of 2.5 and 100 microM Ca2+, whereas effects of peptide hormones (relaxine and EGF) were hardly changed in the presence of 2.5 microM calcium ions, but were markedly inhibited by 100 microM Ca2+. In the EDTA-treated membranes, the AC effects of biogenic amines were reduced, while the effects of peptide hormones were not revealed. On addition of Ca2+, the AC effects of biogenic amines were completely restored, whereas the effects of peptide hormones were not detected or were restored to a non-significant degree. Calmodulin antagonists slightly affected the AC effects of peptide hormones at concentrations efficient in the case of vertebrate AC, but decreased them markedly at higher concentrations. The AC effects of biogenic amines were little sensitive even to high antagonist concentrations. The obtained data show that targets of action of peptide hormones in the infusorian D. anser cell culture are the AC forms whose activity does not D. depends on calcium cations and possibly is regulated by Ca2+/calmodulin, whereas targets of action of biogenic amines are calcium-independent enzyme forms.

[Mating types in the ciliate Dileptus anser. Inheritance and genetic determination].

Hybridological analysis of mating types (MTs) has been first made for the lower ciliate Dileptus anser. Clones of an initially unknown genotype belonging to three MTs (MT I, MT II and MT III), characteristic of D. anser, were isolated from natural reservoirs and further used for crosses. In one group crosses, synclonal inheritance and typical Mendelian behaviour of the character were observed over sexual generations of ciliates. The results suggest that MTs in D. anser may be directly controlled by a single mat locus with three alleles showing peck-order dominance (mat1 > mat2 > mat3). In other words, cells with mat1/mat1, mat1/mat2 and mat1/mat3 genotypes belong to MT I, those with mat2/mat2 and mat2/mat3, and the mat3/mat3 belong to MT II and MT III, respectively. Sexually mature exconjugant clones stably retain their MTs corresponding to their genotypes on vegetative reproduction. The progeny of other group crosses showed various deviations from typical Mendelian behaviour of the character. In some cases, standard Mendelian ratios were more or less violated. Most typical was instability of differentiation for MT in maturing exconjugant clones. Shortly after their maturation, the majority of clones change their MT, rather frequently more than once, although the finally established MT is stably inherited afterwards, during vegetative reproduction. When unstable, exconjugant clones can successively express two or even three MTs characteristic of this species, including MTs that should not have been expected on the basis of parental genotypes available in a given cross. It looks likely that the mat locus in D. anser is complex and multipotential; it is inherited as a whole providing for expression of any MT characteristic of the species (in this respect bearing similarity with Tetrahymena thermophila). Other mechanisms, epigenetic in particular (Nanney, 1958), determine the final expression of one of the three MT potentialities by a given exconjugant clone. Stable, persistent functioning of these mechanisms ensures a stable differentiation for MT and Mendelian behaviour of the character in sexual generations and in crosses. Any disturbances in differentiation control may trigger MT instability in maturing exconjugant clones and violation of regular Mendelian behaviour.

[Inhibitory action of polycationic peptides on hormonal regulation of adenylyl cyclase of the ciliate Dileptus anser].

To analyse molecular mechanisms of regulatory action of different hormones on the activity of the adenylyl cyclase signaling system (ACS) of the ciliate Dileptus anser, we studied the influence on this process of six synthetic polycationic peptides and peptides, corresponding to C-terminal regions of mammalian G-protein 385-394 alphas- and 346-355 alphai2-subunits. As we reported earlier, these peptides block hormonal signal transduction in tissues of the higher eukaryotes. Now it has been found that both polycationic peptides, containing hydrophobic C to-radicals, and branched peptides decrease regulatory effects of peptide hormones (insulin, relaxin) and biogenic amines (serotonin, adrenaline) on adenylyl cyclase (AC) activity and GTP-binding. In regard to the following peptides Cys-epsilonAhx-Trp-Lys-Lys(C10)-Lys2-Lys(C10)-Lys3-Lys(C10)-Tyr-Lys-Lys(C10)-Lys-Lys-amide and [(Gly-Arg-Gly-Asp-Ser-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro- Pro-Gly)2-Lys-EAhx-Cys]2 (epsilonAhx - E-aminocaproyl, C10 - caprinoyl group) their dose-dependent inhibitory action is shown. In cell culture of D. anser with a lower basal AC activity, both hydrophobic and branched peptides stimulated AC and GTP-binding without hormones. The data give evidence that these peptides can activate ACS of ciliates in a receptor-independent manner. No influence of peptides 385-394 alphas and 346-355 alphai2 on hormonal signal transduction in D. anser was observed, due, presumably, to some structural differences of G-proteins of the lower and higher eukaryotes. A conclusion was made about an important role of polycationic regions for functional coupling of hormone-activated receptor and G-proteins in the ciliate D. anser.

[Mating types in the ciliate Dileptus anser. Shortening of maturation period after micrurgical bisection of exconjugant cells]. / Tipy sparivaniia u infuzorii Dileptus anser. Uskorenie polovogo sozrevaniia posle mikrurgicheskoi fragmentatsii ékskon"iugantnykh kletok.

In many ciliates, young exconjugant clones demonstrate sexual immaturity: they are not able to conjugate with mature cells of complementary mating types (MTs). After several scores of cell divisions, a short period of adolescence (partial maturity) commonly occurs followed by maturation, after which these cells are able to conjugate with cells of other, i.e. complementary, MTs again. Tavrovskaja (1981) reported a significant reduction in the maturity period in Dileptus anser clones, grown from exconjugant ciliates regenerated from small cell fragments. To verify this, in the present study exconjugant D. anser cells were bisected with glass needle after 3 to 4 cell divisions following conjugation. The same procedure was performed with survived and regenerated cells on the 2nd and 3rd days. The clones thus obtained were cultivated, and their ability to mate with each of the three standard clones of MT I, II and III was tested week by week. Indeed, in 22 F1 clones from cross N 7C (MT I) x N 2 (MT II) the immaturity period was reduced 1.4-3.4-fold (2.18 in average) after a threefold bisection, as compared with that in intact subclones. Similarly, in 27 F1 clones from cross N 20 (MT I) x N 14 (MT II) this period was shortened 1.6-3.0-fold (2.19 in average). 12 of these clones showed a 0.9-2.4-fold (1.53 in average) reduction after a single bisection, and 1.6-2.8 (2.12 in average) after a threefold operation. Thus, micrurgical fragmentation of young exconjugant cells can be used to accelerate maturation in D. anser.

[Molecular mechanisms of regulatory action of adrenergic receptor agonists on functional activity of adenylyl cyclase signaling system of the ciliates Dileptus anser and Tetrahymena pyriformis]. / Molekuliarnye mekhanizmy reguliatornogo vliianiia agonistov adrenergicheskikh retseptorov na funktsional'nuiu aktivnost' adenilattsiklaznoi signal'noi sistemy infuzorii Dileptus anser i Tetrahymena pyriformis.

Adenylyl cyclase signaling system (ACS) of the higher eukaryotes involves the following main components: receptor, heterotrimeric G protein, adenylyl cyclase (AC), and protein kinase A. At present, these components have been found in cells of different species of the lower eukaryotes. Hence, the signal transduction through ACS of unicellular eukaryotes may have some features in common with those of the higher eukaryotes. We showed earlier that agonists of adrenergic receptors (ARs) regulate AC activity of ciliates Dileptus anser and Tetrahymena pyriformis. The aim of this work was to study molecular mechanisms of AR ligand action on the functional activity of different components of ACS of the ciliates. It has been shown that beta-AR antagonist [3H]-dihydroalprenolol binds membranes of the ciliates with a comparatively lower affinity than those of the higher eukaryotes (Kd for D. anser was 13.4 nM, for T. pyriformis--27 nM). Beta-AR ligands--agonist (-)-isoproterenol and antagonists propranolol and atenolol in competition manner displace [3H]-dihydroalprenolol with IC50 that are 10-100 times higher than corresponding IC50 of beta-AR of the higher eukaryotes. In the presence of GTP, the right shift of competition curves of [3H]-dihydroalprenolol displacement by isoproterenol was obtained, being most considerable in the case of D. anser. Adrenaline and isoproterenol in a dose-dependent manner stimulated GTP-binding in cell cultures of D. anser and T. pyriformis. Suramin (10(-5) M), the inhibitor of heterotrimeric G proteins, completely blocked effects of these hormones. In D. anser culture, adrenaline and isoproterenol in a dose-dependent manner, stimulated AC activity, and its stimulating effects in the presence of beta-AR blockers vanished (propranolol) or decreased to a great extent (atenolol). At the same time the effects were unchanged in the presence of alpha2-AR antagonists yohimbine and idazoxan. These data show the involvement of G protein-coupled beta-AR in signal transduction induced by AR agonists in D. anser cells. In cell culture of T. pyriformis isoproterenol weakly stimulated AC activity, and its effect was completely blocked by beta-AR blockers. Adrenaline in T. pyriformis cells in a dose-dependent manner inhibited AC activity. Inhibiting effect of hormone was decreased in the presence of alpha2-AR blockers. On the basis of the obtained data we concluded that adrenaline in T. pyriformis cells inhibited AC activity through G protein-coupled receptor, being close to alpha2-AR of vertebrate animals.

[Mating types in the ciliate Dileptus anser. Genetical instability in the mating type system]. / Tipy sparivaniia u infuzorii Dileptus anser. Geneticheskaia nestabil'nost' v sisteme tipov sparivaniia.

In F1 and F2 from a cross between two clones of Dileptus anser isolated from natural sources (MT 1 x x MT III; MT = mating type), along with "normal" clones, many clones were observed demonstrating abnormal phenotype with respect to the MT-character. Irregular features of the latter were as follows: a) a delay in maturation; b) temporary reversion to immature or adolescent state, which means instability of maturity state; c) expression of MT I and MT III, rather than MT II as in properly matured clones; d) changes in MT (i.e., MT instability); e) appearance of totally unexpected MTs in terms of the scheme of genetic control of MTs in D. anser previously suggested by Afon'kin and Yudin (1987)--e.g., of all three MTs in F1 from the initial (analysing!) cross. Amazingly, these abnormal D. anser clones closely resembled some selfer-clones of Tetrahymena pigmentosa, previously reported as an example of genetic instability in the ciliate MT system (Simon, 1980; Simon, Orias, 1987).

[Serotypes of the lower ciliate Dileptus anser]. / Serotipy u nizshei infuzorii Dileptus anser.

The author's data on the serotype composition in various clones of Dileptus anser are reviewed. This species is a novel subject of choice for studying the serotype problem in ciliates. The results obtained for serotypes of D. anser demonstrated several features shared also by some higher ciliates of the genera Paramecium and Tetrahymena, i.e. the well known classical models for serotype research. At the same time, some particular data obtained for D. anser do not fit in with the generally accepted picture of relevant events. Special attention is paid to a poor knowledge of the sexual process in this ciliate, which obviously hampers the true understanding of many events. Nevertheless, D. anser may be regarded as a suitable model for studying such important problems of general biology as epigenetic variability or inheritance.

[The hormone-sensitive adenylyl cyclase system of the ciliate Dileptus anser]. / Gormonochuvstvitel'naia adenilattsiklaznaia sistema infuzorii Dileptus anser.

The hormone-sensitive adenylyl cyclase system (AC system) was found and characterized for unicellular eukaryotes--the ciliatae Dileptus anser. It has been first shown that hormones of higher eukaryotes--biogenic amines (adrenalin, isoproterenol and serotonin) and peptide glucagon--stimulate in dose-dependent manner the activity of adenylyl cyclase (AC) of D. anser. The enzymatic activity was stimulated also by guanine nucleotides--GTP and their non-hydrolysable analogue Gpp[NH]p. Stimulating effects of hormones and guanine nucleotides strongly depend on the level of AC basal activity, which is relatively easy to reach (1430 to 3900 pmol cAMP/min per 1 mg of protein). The sensitivity of D. anser AC system to hormones and guanine nucleotides shows the presence of receptor or receptor-related molecules, capable of interacting with the hormone and activating AC through heterotrimeric G-proteins, in ciliatae. On the base of obtained data, a conclusion is made about the similarity of the structural-functional organization of AC systems of D. anser and higher eukaryotes.

[Re-expression of various i-antigens in Dileptus anser after temporary transformation of serotype]. / Vozobnovlenie sovmestnoi ékspressii raznykh i-antigenov infuzoriiami Dileptus anser posle vremennoi transformatsii serotipa.

RNP particles containing 20S prosomes (alpha RNP) isolated from human epidermoid carcinoma cell line A-431 are shown to posses strong and regulated endonuclease activity specific for high-molecular-weight RNA, particularly, specific mRNAs. Furthermore, alpha-RNP destabilize the 3'-untranslated regions of c-myc mRNA, creating a specific cleavage pattern. Cleavage point within Alu sequence in high-molecular-weight RNA has been localized by primer-extension method. This RNase activity is induced under the action of EGF. alpha-RNP involvement in the coordinated control of processing and stability of specific messenger RNA molecules is suggested. The endoribonuclease activity of alpha-RNP can represent a link between EGF signalling pathway and RNA processing and degradation.

[Mode of serotype inheritance in exoconjugant progeny of the ciliate Dileptus anser]. / Nasledovanie serotipov v ékskon''iugantnom potomstve infuzorii Dileptus anser.

Two clones of Dileptus anser, originally isolated from natural reservoirs and referred to below as B and D clones, were found to display different serotypes, when cultured under identical laboratory conditions. On being tested with two different polyclonal rabbit immune sera against each particular clone (the classic immobilization test) these clones showed no cross-reaction. At a standard dilution (1:50) and at a standard exposure time (4 h), either of the two immune sera immobilized 100% or commonly 0% of homologous and heterologous clone cells, respectively. In addition, the difference in serotypes was confirmed by the immunofluorescence analysis. By crossing (conjugation) between B (mating type I) and D (mating type III) cells, exconjugant F1 clones were obtained. Their serotypes were then tested (the same immobilization test) with antisera against both the "parental" clones: some clones were tested before their sexual maturation in ca. one month after conjugation, while others were examined in approximately 4 months after conjugation, i.e. after reaching maturity. Each of the F1 clones could react with both immune sera, which means that they possessed the intermediate, "hybrid" phenotype. Five different F1 clones were selected, and each of them was back-crossed to both "parental" clones, B and D. We succeeded in raising 25 exconjugant F2 (B1, to be more exact) clones from F1 x B crosses and 26 clones from F1 x D crosses. The conventional testing of these clones in 5-10 weeks after conjugation provided quite unexpected results, since among them no segregation for "parental" serotypes was observed. Each of the 51 tested clones demonstrated the "hybrid" serotype--seemingly the same as that of F1 clones. Such a non-Mendelian inheritance of the character is hardly to explain from the standard, canonical assumptions on the genetic control of serotype difference between original "parental" clones (different alleles in one locus? different loci?). Also it does not seem likely that the absence of segregation could result from differential survival of various phenotypes in F2 (although the total viability of exconjugant clones appeared rather low). The above data obviously need further confirmations and experimental analyses. We attempt to discuss the obtained results in terms of the epigene hypothesis (Tchuraev, 1975) and in relation to the epigenetic control of serotype expression in species of the Paramecium aurelia complex and in Tetrahymena thermophila, which are "the chosen few" subjects in ciliate genetics.

[Uracil-DNA glycosidase studied with an immune serum]. / Izuchenie uratsil-DNK-glikozidazy s pomoshch(iu immunnoi syvorotki.

Immune antiserum to uracil-DNA glycosylase was obtained by immunizing rabbits with an enzyme isolated from the rat liver. Antiserum was found to suppress the activity of uracil-DNA glycosylase not only in the extracts of rat liver, but also in the extracts of brain, cardiac muscle, kidney, spleen, thymus of rats, and in those of human placenta too. This enables us to make a conclusion about the similarity in antigenic properties of the enzyme in cells of various types of differentiation. Indirect immunofluorescent test shows a slight staining of the periphery of the nucleus in normal liver hepatocytes and the intensive staining of the inner part of the nucleus in hepatocytes of regenerating liver. Therefore it is concluded that the enzymatic activity increases as cells proliferate. This may be the result of the appearance of uracil in DNA during replication.

[Immunofluorescent study of DNA polymerase alpha in rat cells and tissues]. / Immunofluorestsentnoe issledovanie DNK-polimerazy al'fa v kletkakh i tkaniakh krysy.

An indirect immunofluorescent test based on globulin preparation from a highly specific antiserum against rat liver DNA polymerase alpha was used to direct the enzyme in sections of various tissues of the rat. The immunofluorescent staining was found in cells of the thymus and the wall of intestine crypt, in sparse cells of the intestinal muscular layer, and in cells of the embryo skin epithelium. In sections of liver the intensity of staining and the number of stained cells increased significantly during regeneration. The immunoglobulins did not interact with the cytoplasm and nuclei of skeletal muscle myotubes, with the epithelial cells at the top of intestinal villi, and with erythrocytes. The intracellular localization of the fluorescence observed was of two general types: 1) staining in the region of the nuclear envelope and/or in the cytoplasm; 2) an additional intranuclear staining. The staining of the first type is characteristic of the cells of intact liver and of leyomyocytes. It was also observed in the proliferating cells of thymus and crypt wall, and in cultured myogenic L6 cells. Cells of the embryo skin epithelium, the satellite cells in the skeletal muscle, and about one half of the regenerating liver cells appeared to have the second type of staining. These data serve an indication of possible histotypical differences in in the intracellular localization of DNA polymerase alpha in proliferating cells. It is proposed that the presence of DNA polymerase in resting cells is in association with their ability to respond to the mitogenic stimulus.

[Several enzymic properties of the DNA-polymerase alpha complexes with antibodies]. / Nekotorye fermentativnye svoistva kompleksa DNK-polimerazy al'fa s antitelami.

A highly specific rabbit antiserum against DNA polymerase alpha from regenerating rat liver (antigen AG 1) and an antiserum against the preparation of the enzyme proteolytic fragments possessing catalytic activity (antigen AG 2) were obtained. The enzyme neutralization test revealed that antibodies against AG 2 inhibit the DNA polymerase activity in a much stronger degree, than those against AG 1. Data from a kinetic analysis of the enzyme complexed with the antibodies against AG 1 suggest that the catalytic and binding sites for dNTP and free Mg2+ are altered. The value of apparent Km for activated DNA is unchanged in the DNA polymerase complexes with antibodies both against AG 1 and AG 2.