[Autoradiographic and biochemical studies of protein synthesis in nucleated erythrocytes during the formation of their segregation apparatus]. / Avtoradiograficheskoe i biokhimicheskoe issledovanie sinteza belka v iadernykh éritrotsitakh v period formirovaniia v nikh segregatsionnogo apparata.

It has been shown that during novocaine (4.6 x 10(-3) M) induced formation of the segregation apparatus (SA) the total protein synthesis decreases, the synthesis of certain proteins being characterized by a high rate turnover. In the intact erythrocytes, 1 x 10(-2) cycloheximide (CHM) inhibits the novocaine induced formation of segregation apparatus (SA) and protein synthesis by 90%. The combined action of novocaine and CHM on erythrocytes is accompanied by a decrease in CHM inhibiting effect on protein synthesis. This effect retains for 1.5 h during the action of both compounds. Some vacuoles of SA disposed cytochemically exposed acid phosphatase (AP), which enabled us to consider these as lysosomes. According to AP distribution in lysosomes, they can be classified into 3 groups. In group 1 AP is distributed along the membrane of vacuoles, in group 2 it is associated with the material inside the lysosome, and in group 3 the enzyme is both distributed along the membrane and associated with the material enclosed within the segregation vacuole. Such a mode of AP distribution reflects presumably the functional heterogeneity of lysosomes.

[The effect of oligomycin and cycloheximide on the ultrastructure of the segregation apparatus and on protein synthesis in the erythrocytes of the common frog]. / Vliianie oligomitsina i tsiklogeksimida na ul'trastrukturu segregatsionnogo apparata i sintez belka v éritrotsitakh travianoi liagushki.

The incubation of frog erythrocytes in the Ringer solution with novocaine (4.6 x 10(-3) M) during 24 hours at 10 degrees C provoked vacuole formation (segregation zones). Changes of the novocaine solution for a fresh Ringer solution and the following 48 hour incubation was accompanied by a decrease in the number of vacuoles both electron-translucent and containing membranous material. Simultaneously, the number of vacuoles with amorphous material only and with amorphous and membranous substances was seen to increase. Under the action of cycloheximide (1.10(-2) M) or oligomycin (2.5 x 10(-6) M) on erythrocytes with preformed vacuoles for 48 hours the total number of vacuoles and their dimensions decreased, with numerous amorphous inclusions appearing. Vacuoles with amorphous and membranous material increased in size. Similar ultrastructural changes in the segregation zones under the influence of both the inhibitors were observed showing the appearance of thick threads and a decreased share of electron-translucent vacuoles. A specific effect of cycloheximide, compared to that of oligomycin, involved the expansion of smooth endoplasmic reticulum cisternae. Under the influence of novocaine, 3H-leucin incorporation in proteins of frog erythrocytes was intensified. However, this incorporation was considerably inhibited by cycloheximide. Erythrocytes with segregation zones were more inhibitor susceptible than erythrocytes without vacuoles. The inhibitory effect was stronger early after their administration to the incubation medium, compared to the later periods.

[Electron microscopic and biochemical research on the vacuoles formed in the erythrocytes of frogs as affected by neutral red and novocaine]. / Elektronno-mikroskopicheskoe i biokhimicheskoe issledovanie vakuolei, obrazovavshikhsia v éritrotsitakh liagushek pod vliianiem neitral'nogo krasnogo i novokaina.

No lysosomes were found in the frog intact erythrocytes with electron microscope. Under the influence of neutral red (NR-8.7.10(-5) M) and novocaine (N-4.6.10(-3) M) segregation zones (vacuoles) including these substances are formed. Using electron microscopy and morphometry the action of NR and N for 5 minutes up to 48 hours was found to provoke the formation of four types of vacuoles differing in their morphology: with electron-transparent content, with amorphous inclusions and membrane whorls. The dynamics of vacuole formation, of their changes and amount were followed depending on the time of exposition of these substances. Biochemical investigation of both NR and N isolated vacuoles showed in these some activities of lysosomal marker enzymes--acid phosphatase and N-acetyl-beta,D-glucosaminidase. Ultrastructural investigation of acid phosphatase localization in the isolated vacuoles revealed the histochemical reaction product mainly in electron-translucent vacuoles (primary lysosomes) and partly in electron dense ones (secondary lysosomes). On the ground of the above studies a conclusion is made that in frog erythrocytes treated with NR and N lysosome formation is induced to be followed by the induced autophagocytosis and heterophagocytosis. Some possible ways of the vacuolar system formation in frog erythrocytes and the origin of lysosomal hydrolases are discussed.

[Characteristics of the granules formed in nucleated erythrocytes as affected by cardiotrast]. / Kharakteristika granul, obrazuiushchikhsia v iadernykh éritrotsitakh pod vliianiem kardiotrasta.

Numerous granules are formed in frog erythrocytes under the influence of cardiotrast (C) (diethanolamine-3,5-diiodo-4-pyridone-1-acetic acid). However, as revealed by cytospectrophotometric investigation and X-ray microanalysis, no C was accumulated in these granules. It is known that C can dissociate into diethanolamine and 3,5-diiodo-4-pyridone-1-acetic acid. It was assumed that under the influence of C granule formation may occur at the expense of an uncharged diethanolamine form penetrating into lysosome-like structures to be accumulated by protonation process. Diethanolamine was found to provoke granule formation in frog erythrocytes. It is impossible to reveal substance in granules because it is colourless and has no ultraviolet absorbtion band. Under the influence of some inhibitors of energy metabolism on granule formation and the granules formed, their inhibitory effect is exerted on the process of granule formation. In granules isolated by differential centrifugation activity of some marker lysosomal enzymes was found which enabled us to attribute these granules to lysosome-like structures.

[Effect of metabolic inhibitors on formed novocaine and neutral red segregation zones in frog erythrocytes]. / Vliianie ingibitorov metabolizma na sformirovannye zony segregatsii novokaina i neitral'nogo krasnogo v éritrotsitakh liagushki.

Effects of some metabolic inhibitors, as well as of biologically active compounds (diakarb, ethidium bromide and a phenanthridine alkaloid sanguinarine) on the formed novocaine and neutral red segregation zones were studied. The volume of granules diminished under the influence of a glycolytic inhibitor iodoacetate, uncouplers of oxidative phosphorylation (2,4-dinitrophenol and carbonyl cyanide trifluoromethoxyphenylhydrozone), and respiratory inhibitors (antimycin A and rotenone), as well as under the influence of cycloheximide - an inhibitor of protein synthesis. Diakarb, ethidium bromide or sanguinarine also provoked a regression of the segregation zones. It has been found that all these compounds are inhibitors of ATPase activity of the isolated segregation zones. A possible mechanism of volume decreasing in segregation zones under the influence of both the metabolic inhibitors and diakarb, ethidium bromide and sanguinarine is discussed.

[ATPase activity of the novocaine segregation zones isolated from the erythrocytes of the common frog]. / ATFaznaia aktivnost' zon segregatsii novokaina, izolirovannykh iz éritrotsitov travianoi liagushki.

Novocaine segregation zones in frog's erythrocytes, isolated by differential centrifugation, were shown to be ATPase active. The enzyme displays half of its maximum activity at 0.18 Mm ATP concentration to be inhibited by high concentrations of ATP. ATPase is activated by both Mg2+ and Ca2+ (in a lesser degree), with the maximum activity being at pH 7.5. A 5 minutes heating without the substrate results in decreasing the enzyme activity at 30 degrees, and in the total inhibition at 50 degrees C. Along with ATP, the enzyme can hydrolyse GTP and, in a lesser degree, ADP and sodium pyrophosphate. The ATPase activity is not effected with oligomycin (0.5-1.5 mkg/ml) or ouabaine (0.1 mM). Oligomycin in concentration 5 micrograms/ml induced non-specific inhibition of ATPase. Uncouplers, like 2,4-dinitrophenol and carbonyl cyanid p-trifluorometoxyphenylhydrazone, stimulate the enzyme activity. The lack in the ATP-ase sensitivity to oligomycin (specific inhibitor of mitochondrial F1-ATPase) and ouabaine (specific inhibitor of Na+, K+-ATPase) may suggest that the ATPase activity of novocaine segregation zones in frog's erythrocytes is not associated with a random contamination with mitochondria or cytoplasmic membranes. The ATPase under study has much in common with the lysosomal +H-ATPase. The results obtained support a hypothesis that +H-ATPase may function as a course of protones for maintaining acidic medium in segregation zones and promote accumulation of weak bases by means of their protonation.

[Cytophotometric study of the granules formed in frog erythrocytes incubated in solutions of acridine orange, chloroquine and daunorubicin]. / Tsitofotometricheskoe issledovanie granul, obrazuiushchikhsia v éritrotsitakh liagushki pri inkubatsii ikh v rastvorakh akridinovogo oranzhevogo, khlorokhina i daunorubitsina.

Granules arising in the cytoplasm of Rana temporaria erythrocytes incubated in either solution containing acridine orange, chloroquine or antibiotic daunorubicine were studied cytophotometrically. The stuff concentrations in the granules were estimated under various conditions of incubation. The stuff concentrations in the granules appeared to be 1000-fold higher than in the incubation solutions. The average concentration in the granules did not depend on either the initial concentration of the stuff solution or the incubation time. The stuff concentration in the granules decreases with the increase of the granule size. Mechanisms of cation-acid segregation and accumulation by living cell are discussed.

[Effect of inhibitors of energy metabolism and protein synthesis on the process of neutral red segregation in frog erythrocytes]. / Vliianie ingibitorov énergeticheskogo metabolizma i sinteza belka na protsess segregatsii neitral'nogo krasnogo v éritrotsitakh liagushki.

Effects of inhibitors of energy metabolism and protein synthesis on Neutral red segregation in frog erythrocytes were studied. Inhibitors of both glycolysis and respiration significantly reduced formation of segregation zones. This influence was most striking with antimycin A, rotenone and cyanide. This indicates that intact respiratory pathways may play an important part in the process of Neutral red segregation. Such uncouplers as FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) and 2,4-dinitrophenol (DNP) as well as inhibitors of oxidative phosphorylation (arsenate and azide) are also very effective in inhibiting the Neutral red segregation at low concentrations. The effects of these uncouplers and of olygomycin suggest an important role of ATP as an energy source for the segregation process. An inhibitor of protein synthesis, such as cycloheximide, produces some reduction in segregation zones formation. Trapping of Neutral red by protonation could readily explain the high level of this dye accumulation in nucleated erythrocytes. The fact that low concentrations of FCCP and DNP inhibit the process of segregation brings a supporting evidence for the possibility of the ATP-driven proton pump involved in Neutral red segregation.

[A cytophotometric study of granules forming in frog erythrocytes during incubation in solutions of neutral red and novocaine]. / Tsitofotometricheskoe issledovanie granul, obrazuiushchikhsia v éritrotsitakh liagushki pri inkubatsii ikh v rastvorakh neitral'nogo krasnogo i novokaina

Granules arising in the cytoplasm of Rana temporaria erythrocytes incubated in solutions containing either a stain (neutral red) or an alcaloid (novocaini) were studied cytophotometrically. The stuff concentrations in the granules were estimated under various conditions of incubations. The stuff concentrations in the granules appeared to be dozens- or hundreds--fold higher than in the incubation solutions. The average concentration in the granules did not depend either on the initial concentration of the stuff solution, or on the incubation time or on the volume of the incubation solution. The stuff concentration in the granule decreases with the increase in the granule size.