[Study of the binding of nuclear proteins from Plasmodium berghei strains with different chloroquine sensitivity to oligonucleotides corresponding to regulatory elements of multidrug resistance (mdr1) gene].

Using electrophoretic mobility shift assay we first revealed in the nuclear extracts of the rodent malaria parasite Plasmodium berghei (P. berghei) proteins, which bind specifically to the double-stranded oligonucleotides reproducing the binding sites of the transcription factors of AP1 family, NF-IL6 and SP1 involved in the up-regulation of human multidrug resistance (mdr1) gene and to the oligonucleotide corresponding to the element responsive for the stimulation by serum (SRE). The nuclear proteins isolated from the P. berghei strains with various chloroquine sensitivity bound differently to the most of the oligonucleotide probes used. Mutations in the consensus sequences of AP1, NF-IL6 and SRE led to the loss of some DNA-protein complexes, suggesting the existence of malaria parasite nuclear proteins, whose DNA-binding domains are similar to DNA-binding domains of NF-IL6, SRF1, and AP1 family members. These proteins exhibit greater activities in chloroquine resistant strains. The results obtained denote profound alterations in the plasmodium regulatory apparatus occurred as the result of selection on chloroquine resistance.

Differences in binding of nuclear proteins from P. berghei strains differing by chloroquine resistance to oligonucleotides corresponding to mdr1 gene regulatory elements.

Proteins specifically reacting with AP1, MEF1, NF-IL6, and SP1 transcription factor binding sites were detected for the first time in nuclear extract of P. berghei (rodent malaria parasite) using gel retardation assay. P. berghei strains with different chloroquine resistance exhibited appreciable differences in the pattern of nuclear protein binding to the majority of the studied sites, which attests to changes in the plasmodial regulatory system during chloroquine resistance selection.

[Culture of Plasmodium berghei with the short life span in mouse erythrocytes: evaluation of chloroquine resistance in three strains of the malaria parasite]. / Korotkozhivushchaia kul'tura Plasmodium berghei v éritrotsitakh myshei: opredelenie ustoichivosti k khlorokhinu u trekh shtammov maliariinogo parazita.

This comparative study was made to examine chroloquine inhibition of C14-hypoxanthine incorporation (preferably nucleic acid precursor for plasmodium) in the short-living red blood cell cultures in mice infected with P. berghei strains differently sensitive to the agent 24 hours after incubation. These included 1) chloroquine-sensitive H strain; 2) LNK-65 strain having a spontaneously 2-3-fold decreased sensitivity to the agent; 3) strain LNK-65 ChR selected for high resistance to chloroquine. IC50 (chloroquine concentrations that ensure 50% inhibition of the incorporation of nucleic acid precursor into the cells of the parasite) was estimated for 3 P. berghei strains that were differently sensitive to chloroquine. These in vitro values adequately reflect the sensitivity of these strains to the agent in vivo.

[A comparative restriction analysis of the DNA of strains of the malarial parasite sensitive and resistant to chloroquine]. / Sravnitel'nyi restriktsionnyi analiz DNK shtammov maliariinogo parazita chuvstvitel'nogo i rezistentnykh k khlorokhinu.

A comparative restriction analysis was made for DNA in malaria parasites, strain H sensitive to chloroquinone, strain LNK-65 with spontaneously occurred resistance to the agent, and breeding strain LNK-65 ChlR highly resistant to it. DNA hydrolysis with EcoR1, HindIII, and BamH1 endonucleases revealed permanent differences in the DNA restriction pattern of malaria parasites. There were additional restriction bands as part of DNA restricts in the strain LNK-65 Chl bred from LNK-65 for high resistance to chloroquine on EcoR1-, HindIII-, and BamH1-hydrolysis. Great differences in the DNA restriction pattern in the strains H and LNK-65 are likely to be due to their belonging to various strains, such as P.berghei and P.yoelii, respectively. Comparison of the DNA restriction pattern of the host (murine leukocytes) and the malaria parasite suggests the plasmodium DNA is adequately removed from the host DNA.

Chloroquine resistance of Plasmodium berghei: biochemical basis and countermeasures.

Microsomal monooxygenases, enzymes that metabolize xenobiotics, may be responsible for the chloroquine resistance of malarial parasites. Plasmodium cells contain cytochrome P-450 and exhibit aryl hydrocarbon hydroxylase and aminopyrine N-dimethylase activity, two monooxygenases that inactivate chloroquine. The activities of these monooxygenases are considerably higher in chloroquine-resistant strains of Plasmodium berghei than in the chloroquine-sensitive strain of the parasite. Inhibitors of microsomal monooxygenases have the potential to overcome the chloroquine resistance of Plasmodium spp., and, of those inhibitors tested, the copper-lysine complex, copper(lysine)(2), was the most effective.

[Microsomal monooxygenase inhibitors as promising agents for overcoming the drug resistance of the malaria parasite]. / Ingibitory mikrosomal'nykh monooksigenaz kak perspektivnye sredstva preodoleniia lekarstvennoi ustoichivosti u maliariinogo parazita.

A relationship was found between resistance of malarial plasmodium to chloroquine and the increased activity of microsomal monooxygenases, metabolizing drugs in the parasite. A search for effective inhibitors of the enzymatic system was initiated. For this purpose inhibitory effects of 17 alpha-hydrodeoxycorticosterone (substance S), 21-acetate-17 alpha-hydroxydeoxycorticosterone (acetate of substance S), 4-bromomethyl-2,2,5,5-tetramethyl-3-imidazoline-3-oxide-1-oxyl (RBr), Cu(lysine)2 on activity of arylhydroxycarbone hydroxylase were studied using mice liver microsomes and homogenate of mice malaria cells Plasmodium berghei. Cu(lysine)2 and phenylhydrazine were found to be the most effective inhibitors of the enzyme in samples containing mice liver microsomes or malarial parasite. The data obtained suggest that the inhibitors of microsomal monooxygenases may serve as means for a decrease in malarial parasite resistance to chloroquine.

[Role of histamine as an intermediary in the action of estradiol on the rat uterus: inhibition of the hormonal induction of enzymes using histamine antagonists]. / Rol' gistamina kak posrednika v deistvii éstradiola ma matku krys: tormozhenie gormonal'noi induktsii fermentov s pomoshch'iu antagonistov histamina.

The administration of methiamide, a blocker of histamine H2-receptors, to rats was shown to prevent the induction of hexokinase (HK) and glucose-6-phosphate dehydrogenase (G-6PD) caused by both histamine and estradiol. An inhibitory effect of methiamide remained stable up to 16 h after histamine and estradiol administration. Diphenhydramine, a blocker of histamine H1-receptors, produces a short-term inhibitory effect on the HK and G-6PD activity stimulated by histamine and estradiol. The short-term nature of the diphenhydramine effect may be attributed to a low affinity to histamine H2-receptors and histamine fast dissociation. It may be concluded that some of the estradiol effects, particularly the HK and G-6PD induction in the uterus are histamine-mediated, apparently by means of H2-receptors. The administration of histamine to rats increases the uterus mass causing its inundation, and H1- and H2-blockers prevent this effect of histamine. However, the above histamine antagonists do not inhibit uterus inundation caused by estradiol. A conclusion is made that estradiol stimulates the uterus inundation not only by means of histamine but also by some other means.

[Higher microsomal monooxygenase activity in chloroquine-resistant strains of malarial plasmodium as a possible cause of drug stability]. / Povyshennaia aktivnost' mikrosomal'nykh monooksigenaz khlorokhinorezistentnykh shtammov maliariinogo plazmodiia kak veroiatnaia prichina lekarstvennoi ustoichivosti.

Ability to hydroxylate benz(alpha)pyrene--a substrate of arylhydrocarbone hydroxylase (AHH) was distinctly increased in lysates of erythrocytes containing malarial plasmodium. Hydroxylation of benz(alpha)pyrene was inhibited by methyrapone--an inhibitor of microsomal monooxygenases. Activity of AHH was increased from 2- to 3-fold in chloroquine-resistant plasmodium strains as compared with the drug-sensitive strains. Resistance of Plasmodium berghei to chloroquine appears to involve an activation of the monooxygenases system in the parasite.

Biochemical cascade mediating the estradiol action.

The data suggest that estradiol enhances the formation of histamine in rat uterus by induction of histidine decarboxylase; histamine activates adenylate cyclase providing accumulation of cyclic 3',5'-AMP, which, probably, induces glycolytic enzymes via phosphorylation of chromatin proteins, and mediates other estradiol effects. The chain of successively acting enzymes and mediators constitutes, obviously, a cascade amplifying the estradiol action. Since histamine is known to act as an intercellular mediator, attempts were made to find out the distribution of estradiol, histamine, and cyclic 3',5'-AMP among uterine cells. Autoradiography has shown that 3H-estradiol is bound by the nuclei of myometrium cells, 3H-histamine was found in the cytoplasm of these cells, 3H-cyclic 3',5'-AMP is selectively bound by the cells of capillary endothelium of the uterus. The estradiol mediators seem to spread their effect on different types of cells which form together a kind of a multicellular functional system.

[Role of cAMP-dependent phosphorylation of rat uterus nuclear proteins in the mechanism of estradiol action]. / Rol' tsAMF-zavisimogo fosforilirovaniia iadernykh belkov matki krys v mekhanizme deistviia éstradiola.

In experiments on ovariectomized rats cAMP-dependent phosphorylation of the uterine nuclear tissue proteins under the action of estradiol, histamine and cAMP was studied. It was shown, that phosphorylation of the uterine isolated nuclear proteins under the influence of endogenous proteinkinases increases 6 hours after estradiol, histamine and/or cAMP injection to the animals. Proteinkinase activity of the uterine tissue nuclei are higher, when exogenous histone H1 is used as a substrate. The ability of histone H1, isolated from the uterine tissue 6 hours after estradiol, histamine or cAMP injection, to be phosphorylated in vitro with exogenous cAMP-dependent proteinkinase is lowered, that is likely to be resultant of preceding phosphorylation of these proteins in vivo.

Multistage functional system amplifying and spreading the effect of estradiol in rat uterus.

Estradiol is demonstrated to induce histidine decarboxylase, and histamine is shown to activate adenylate cyclase in rat uterus. Histamine and cyclic 3',5'-AMP mimic the effects of estradiol in that they enhance RNA synthesis, induce glycolytic enzymes and uterus imbibition. The data suggest that estradiol enhances by induction of histidine decarboxylase the formation of histamine, the latter activates adenylate cyclase providing accumulation of cyclic 3',5'-AMP, which, probably, induces glycolytic enzymes through phosphorylation of chromatin proteins, and mediates other estradiol effects. The chain of successively acting enzymes and mediators constitutes, obviously, a cascade amplifying estradiol action. Since histamine is known to act as an intercellular mediator, attempts were made to find out the distribution of estradiol histamine and cyclic 3',5'-AMP among uterus cells. Autoradiography has shown that [3H]-estradiol is bound by the nuclei of myometrium cells, [3H]-histamine was found above the cytoplasm of these cells, E13H]-cyclic 3',5'-AMP is selectively bound by the cells of capillary endothelium of the uterus. The estradiol mediators seem to spread effect of hormone on cells of different types which form together a kind of multicellular functional system.

[Multiphasic regulatory system mediating the effect of estradiol in rat uterus]. / Mnogostupenchataia reguliatornaia sistema, oposreduiushchaia deistvie estradiola v matke krys.

Estradiol is shown to induce histidine decarboxylase and histamine to activate adenylate cyclase in the rat uterus. Cyclic AMP like histamine simulates the effect of estradiol, intensifying RNA synthesis and inducing glycolytic enzymes and uterus inhibition. It was found by autoradiography that 3H-estradiol is accepted by the nuclei of some myometrium cells, 3H-histamine by their cytoplasm and 3H-cAMP is selectively bound by endothelium cells of the uterus capillaries. The estradiol messengers (histamine and cAMP) seem to mediate hormonal effect of some uterus heterofunctional cells forming a kind of multicellular functional system.