Vol 89, No 6 (2024)

Currently, numerous associations between genetic polymorphisms and various diseases have been characterized through Genome-Wide Association Studies. The majority of clinically significant polymorphisms are localized in non-coding regions of the genome. While modern bioinformatic resources make it possible to predict molecular mechanisms that explain the influence of non-coding polymorphisms on gene expression, such hypotheses require experimental verification. This review discusses the methods for elucidating the molecular mechanisms underlying the dependence of disease pathogenesis on specific genetic variants within non-coding sequences. A particular focus is on the methods to identify the transcription factors with binding efficiency contingent upon polymorphic variations. Despite remarkable progress in the bioinformatic resources enabling the prediction of the impact of polymorphisms on disease pathogenesis, the imperative for experimental approaches to this inquiry still persists.

Tyrosine hydroxylase (TH) catalyzes hydroxylation of L-tyrosine to L-3,4-dihydroxyphenylalanine, the initial and rate-limiting step in the synthesis of dopamine, noradrenaline, and adrenaline. Mutations in the human TH gene are associated with hereditary motor disorders. The common C886T mutation identified in the mouse Th gene results in the R278H substitution in the enzyme molecule. We investigated the impact of this mutation on the TH activity in the mouse midbrain. The TH activity in the midbrain of Mus musculus castaneus (CAST) mice homozygous for the 886C allele was higher compared to C57BL/6 and DBA/2 mice homozygous for the 886T allele. Notably, this difference in the enzyme activity was not associated with changes in the Th gene mRNA levels and TH protein content. Analysis of the TH activity in the midbrain in mice from the F2 population obtained by crossbreeding of C57BL/6 and CAST mice revealed that the 886C allele is associated with a high TH activity. Moreover, this allele showed complete dominance over the 886T allele. However, the C886T mutation did not affect the levels of TH protein in the midbrain. These findings demonstrate that the C886T mutation is a major genetic factor determining the activity of TH in the midbrain of common laboratory mouse strains. Moreover, it represents the first common spontaneous mutation in the mouse Th gene whose influence on the enzyme activity has been demonstrated. These results will help to understand the role of TH in the development of adaptive and pathological behavior, elucidate molecular mechanisms regulating the activity of TH, and explore pharmacological agents for modulating its function.

The astrocytic NMDA receptor is a heterotetramer consisting of 7 different subunits. Receptor expression and properties are largely determined by its subunit composition. Astrocytic NMDA receptors have their own functional features – they are weakly sensitive to magnesium ions and have low calcium permeability. Activation of astrocytic NMDA receptors plays an important role in regulating intracellular processes such as gene expression and mitochondrial function. NMDA receptors are involved in astrocytic calcium signaling and can be activated by both ionotropic and metabotropic pathways. Astrocytic NMDA receptors are involved in neuroglial interactions, affecting synaptic plasticity. They are also involved in astrovascular interactions and play a role in regulating vascular tone. Astrocytic NMDA receptors participate in pathological conditions such as ischemia and hyperammonemia. Their blockade prevents negative changes in astrocytes in these pathologies.

The work presents the results of an in vitro and in silico study of the formation of amyloid-like structures under harsh denaturing conditions by the nonspecific OmpF porin of Yersinia pseudotuberculosis (YpOmpF), a membrane protein with a β-barrel conformation. It has been shown that in order to obtain amyloid-like porin aggregates, preliminary destabilization of its structure in a buffer solution with an acidic pH value at elevated temperature, followed by long-term incubation at room temperature is necessary. After heating at 95 °C in a solution with pH 4.5, significant conformational rearrangements are observed in the porin molecule at the level of the tertiary and secondary structure of the protein, which are accompanied by an increase in the content of the total β-structure and a sharp decrease in the value of the characteristic viscosity of the protein solution. Subsequent long-term exposure of the resulting unstable intermediate YpOmpF at room temperature leads to the formation of porin aggregates of various shapes and sizes that bind thioflavin T, a specific fluorescent dye for the detection of amyloid-like protein structures. Compared to the initial protein, early intermediates of the amyloidogenic porin pathway, oligomers, have been shown to have increased toxicity to Neuro-2aCCL-131™ mouse neuroblastoma cells. The results of computer modeling and analysis of changes in intrinsic fluorescence during protein aggregation suggest that during the formation of amyloid-like aggregates, changes in the structure of YpOmpF affect not only areas with an internally disordered structure corresponding to the external loops of the porin, but also the main framework of the molecule, which has a rigid spatial structure inherent to β-barrel.

In the Institute of cytology and genetics (Novosibirsk) for over 85 generations takes place a selection of grey rats for high aggression toward humans (aggressive rats) or its complete absence (tame rats). Aggressive rats are an interesting model to study fear-induced aggression. Benzopentathiepin TC-2153 exerts an antiaggressive effect on aggressive rats and affects serotonergic system – an important regulator of aggression. The aim of this study was to investigate the TC-2153 effect on key serotonergic system enzymes – tryptophan hydroxylaze 2 (TPH2) and monoamine oxydase A (MAOA) – in the brain of aggressive and tame rats. TC-2153 (10 or 20 mg/kg) or vehicle were administered once i.p. to male aggressive and tame rats. TPH2 and MAOA enzymatic activity, mRNA and protein levels were assessed. Selection for high aggression level resulted in elevated Tph2 mRNA levels in the midbrain, TPH2 protein in hippocampus and TPH2 and MAOA proteins in hypothalamus. MAO activity was higher in the midbrain and hippocampus of aggressive rats while TPH2 activity did not differ between the strains. Single TC-2153 administration decreased TPH2 and MAO activity in hypothalamus and midbrain respectively. The drug acted upon MAOA protein levels in hypothalamus: elevated that of aggressive rats and decreased in the tame ones. Thus, this study shows profound differences in the expression and activity of the key serotonergic system enzymes in the brain of rats selectively bred for highly aggressive behavior toward humans and its absence, and effects of benzopentathiepin TC-2153 on these enzymes may point to the mechanisms of its antiaggressive action.

Primary excitation energy transfer and charge separation reactions in photosystem I (PSI) from the extremophile desert green alga Chlorella ohadii, grown in low light, were studied using broadband femtosecond pump-probe spectroscopy in the spectral range from 400 to 850 nm and in the time range of 50 fs–500 ps. Photochemical reactions were induced by the excitation into the blue and red edges of the chlorophyll Qy absorption band, and compared with similar processes in PSI from the cyanobacterium Synechocystis sp. PCC 6803. When PSI from C. ohadii was excited at a wavelength of 660 nm, the processes of energy redistribution in the light-harvesting antenna of the complex were observed in a time interval of up to 25 ps, while the formation of a stable ion-radical pair P₇₀₀+A₁− was kinetically heterogeneous with characteristic times of 25 and 120 ps. With an alternative variant of excitation into the red edge of the Qy band at a wavelength of 715 nm, in half of the complexes, primary charge separation reactions were observed in the time range of 7 ps. In the rest of the complexes, the formation of the ion-radical pair P₇₀₀+A₁− was limited by energy transfer and occurred with a characteristic time of 70 ps. Similar photochemical reactions in PSI from Synechocystis 6803 were significantly faster: upon excitation at a wavelength of 680 nm, in ~30% of the complexes, the formation of primary ion-radical pairs occurred with a time of 3 ps. Upon excitation at 720 nm, kinetically unresolvable ultrafast primary charge separation was observed in half of the complexes, and the subsequent formation of a P₇₀₀+A₁− ion-radical pair was observed at 25 ps. The photodynamics of PSI from C. ohadii had a noticeable similarity with the processes of excitation energy transfer and charge separation in PSI from the microalga Chlamydomonas reinhardtii; however, in the PSI from C. ohadii slower components in the energy transfer dynamics were also observed.