Cyclophosphamide metabolite inducing apoptosis in RLS mouse lymphosarcoma cells is a substrate for P-glycoprotein.

RLS lymphosarcoma characterized by enhanced expression of mdr1a and mdr1b genes encoding P-glycoprotein is insensitive to low doses of cyclophosphamide, but is susceptible to its high doses approximating the maximum tolerated doses. Induction of apoptotic death of RLS cells by high doses of cyclophosphamide was demonstrated by cytofluorometry and electrophoresis. Experiments on RLS(40) tumor cells derived from RLS lymphosarcoma and characterized by more intensive expression of mdr1a/1b genes showed that the therapeutic effects of cyclophosphamide increased under conditions of simultaneous suppression of these genes by specific small interfering RNA (siRNA). These findings suggest that active cyclophosphamide metabolite can be a substrate for P-glycoprotein.

[Recognition of the potential SF-1 binding sites by SiteGA method, their experimental verification and search for new SF-1 target genes].

The SF-1 (Steroidogenic Factor-1) is a transcription factor known as a key regulator of the steroidogenic gene expression. SF-1 is required for the development and functioning at all levels of the hypothalamic-pituitary-gonadal and adrenal axis. Also it plays an essential role in sex determination. SF-1 is a member of the nuclear receptor superfamily and it activates gene expression by binding to DNA in a monomeric form. Here, we report the results of potential SF-1 binding sites identification by using the SiteGA recognition method. The SiteGA method was implemented using a genetic algorithm (GA) involving a iterative discriminant analyses of local dinucleotide context characteristics. These characteristics were compiled not only over the core binding sites region but over its flanks as well. Developed SiteGA method is characterized by considerably better recognition accuracy when compared to that for the weight matrix method. The experimental tests demonstrated that 83% of the sites recognized by the SiteGA method in the regulatory regions of steroidogenic genes, indeed, interact with the SF-1 factor. We also estimated the density of predicted sites in regulatory region of genes, the members of different functional groups and developed the criterion to search for new SF-1 target genes in genome sequences.

Possible molecular-cellular mechanisms of the regulation of gene expression during learning.

This study is an analysis of the regulatory mechanisms of plasticity. The first part provides a short review of the role of DNA-binding transcription factors in possible regulatory pathways and their activity in the mechanisms of plasticity. Our own data obtained in studies of the molecular mechanisms underlying the formation of conditioned defensive reflexes in Helix are then presented. These studies show that formation of defensive types of plasticity in Helix is accompanied by serotonin-induced translocation of a protein with Rf 0.58 and increases in G-protein activity, protein kinase A activity, and expression of the c-fos gene. Transcription factors CRE and AP-1 probably have roles in the learning process. Gel shift assays demonstrated the existence of transcription factors of the CRE and AP-1 families in adult snails. In juvenile snails, which were unable to form defensive types of plasticity, the serotonin protein with Rf 0.58 (the learning "marker") was absent from and was not induced in the CNS. Gel shift assay results also showed that transcription factors of the AP-1 family were not present and were not induced by serotonin or the protein kinase A activator forskolin, though these snails had significant levels of CRE transcription factors. Serotonin and forskolin increased the DNA-binding activity of CRE in juvenile Helix. The lack of activity of transcription factors of the AP-1 family in juvenile snails may explain their inability to development sensitization and conditioned defensive reflexes.

[Statistical study of the nucleotide sequence of a prokaryotic promoter. Structural elements determining the efficacy of the transcription initiation stages]. / Statisticheskoe issledovanie nukleotidnoi posledovatel'nosti prokarioticheskogo promotora. Elementy struktury, opredeliaiushchie éffektivnost' protekaniia étapov nachala transkriptsii.

Nucleotide sequences of 188 promoter-containing DNA regions have been studied by the computer statistic analysis. Undecanucleotide NTT(G/C)TTGACA(A/T) or (G/C) X TT(G/C)A(G/C)A(A/T)TT(G/T) (recognition site) and heptanucleotide RTATATR or TATAATR (initiation site) separated by 12-19 base pairs are characteristic of a "generalized" promoter structure. Promoters can function if a minimal level of correspondence for their recognition and initiation sites to a generalized structure is attained (the correspondence function value for the whole structure is not lower than 0,61; for the most effective promoters it may be equal to 1). The transcription start is situated 3-9 base pairs after initiation site, 4-7 pairs distance being the most effective. Transcription can start from any nucleotide, preferably with A or G. The start from A is the most effective if it is contained within the CAC or CAT trinucleotides. The promoter efficiency is enhanced by some additional structural factors: the presence of an extended A-T rich region directly before the recognition site; availability of integral promoter structures or several RNA polymerase binding sites in the preceding nucleotide sequence. A characteristic feature of the promoter is the presence of either the dyadic axial symmetry elements in the initiation and recognition sites as well as in the intermediate region, or the A-T rich area in the latter.

[Possible molecular and cellular mechanisms of gene expression regulation during learning]. / Vozmozhnye molekuliarno-kletochnye mekhanizmy reguliatsii ékspressii genov pri obuchenii.

The learning plasticity was shown to be accompanied by a serotonin-induced translocation of Rf 0.58 protein, increasing of the G-protein activity, as well as PKA and c-fos gene expression in Helix. The learning marker Rf 0.58 is absent from the juvenile snails. The AP-1 transcription factors in them cannot be determined or induced with serotonin. The level of CRE factors in juvenile snails was comparable with adult and can be regulated by serotonin. The impossibility of sensitisation and avoidance conditioning reflex in juvenile snails can be explain with the AP-1 factors deficit.

Elevated basal activity of tryptophan oxygenase in alcohol-preferring C57BL mice.

Tryptophan oxygenase activity in alcohol-preferring C57Bl mice and control CBA and DBA/2 mice was studied under nonstressful conditions and after glucocorticoid-induced stress. Elevated basal tryptophan oxygenase activity in C57Bl mice is probably responsible for reduced brain content of tryptophan and serotonin associated with alcohol preference.

Predisposition to alcoholism, tryptophan oxygenase activity, and structure of intron 6 in the corresponding gene of C57BL/6J, CC57BR/Mv, and BALB/cJ mice.

Crossbred CC57BR/Mv mice inherited tryptophan oxygenase gene and predisposition to alcohol consumption from parent BALB/c and C57BL mice, respectively. In CC57BR/Mv mice no relationships were found between alcohol consumption, tryptophan oxygenase activity, and single nucleotide substitutions in intron 6 of the TDO2 gene associated with predisposition to alcoholism in humans.