Biochemistry (Moscow)

Selective labeling of barstar by the stable 15N isotope of the valine residue with high selectivity of the label incorporation resulting from the process of gene expression in Escherichia coli BL21(DE3) has been optimized. We have shown that α-aminooxyacetic acid (AOAA) significantly reduces the isotope redistribution, thus increasing the selectivity of 15N incorporation into the synthesized protein, as detected by 2D-NMR. Quantitative measurements were used to determine the selectivity for the incorporation of isotope-labeled valine residue, which was 96% in the case using AOAA. Studies of the dynamics of barstar synthesis showed that no suppression of barstar yield is observed under the regulation of the T7 polymerase expression system by isopropylthio-β-D-galactoside (IPTG) and rifampicin using AOAA.

The review discusses the data on vitamin D accumulation in animals, plants, and other organisms. 7-Dehydrocholesterol (7-DHC) and ergosterol are considered to be the only true precursors of vitamin D, although even vitamin D2 (ergocalciferol) is not fully comparable to vitamin D3 (cholecalciferol) in regard to their functions. These precursors are converted by UV radiation into the corresponding D vitamins. There are a few published reports that this reaction can also occur in the dark or under blue light, which is unexpected and requires explanation. Another unexpected result is conversion of pro-vitamins D (7-DHC and ergosterol) into vitamin D3 and D2 via pre-vitamin D at low temperatures (<16°C) in the lichen Cladonia rangiferina. An extensive survey of literature data leads to the conclusion that vitamin D is synthesized from (1) 7-DHC via lanosterol (D3) in land animals; (2) 7-DHC via cycloartenol (D3) in plants; (3) ergosterol via lanosterol (D2) in fungi; and (4) 7-DHC or ergosterol (D3 or D2) in algae. Vitamin D primarily accumulates in organisms, in which it acts as a pro-hormone, e.g., land animals. It can also be found as a degradation product in many other species, in which spontaneous conversion of some membrane sterols upon UV irradiation leads to the formation of vitamins D3 or D2, even if they are not necessarily needed by the organism. Such products accumulate due to the absence of metabolizing enzymes, e.g., in algae, fungi, or lichens. Other organisms (e.g., zooplankton and fish) receive vitamins D with food; in this case, vitamins D do not seem to carry out biological functions; they are not metabolized but stored in cells. A few exceptions were found: the rainbow trout and at least four plant species that accumulate active hormone calcitriol (but not vitamin D) in relatively high amounts. As a result, these plants are very toxic for grazing animals (cause enzootic calcinosis). In connection with the proposal of some scientists to produce large quantities of vitamin D with the help of plants, the accumulation of calcitriol in some plants is discussed.

The interaction between duodenase, a newly recognized serine proteinase belonging to the small group of Janusfaced proteinases, and α1-proteinase inhibitor (α1-PI) from human serum was investigated. The stoichiometry of the inhibition was 1.2 mol/mol. The presence of a stable enzyme–inhibitor complex was shown by SDS-PAGE. The mechanism of interaction between duodenase and α1-PI was shown to be of the suicide type. The equilibrium and inhibition constants are 13 ± 3 nM and (1.9 ± 0.3)·105 M–1·sec–1, respectively. Based on the association rate constant of the enzyme–inhibitor complex and localization of duodenase and α1-PI in identical compartments, α1-PI is suggested to be a duodenase inhibitor in vivo.

The binding of ADP and ATP to noncatalytic sites of dithiothreitol-modified chloroplast ATP synthase was studied. Selective binding of nucleotides to noncatalytic sites was provided by preliminary light incubation of thylakoid membranes with [14C]ADP followed by its dissociation from catalytic sites during dark ATP hydrolysis stimulated by bisulfite ions (“cold chase”). Incorporation of labeled nucleotides increased with increasing light intensity. Concentration-dependent equilibrium between free and bound nucleotides was achieved within 2–10 min with the following characteristic parameters: the maximal value of nucleotide incorporation was 1.5 nmol/mg of chlorophyll, and the dissociation constant was 1.5 µM. The dependence of nucleotide incorporation on Mg2+ concentration was slight and changed insignificantly upon substituting Ca2+ for Mg2+. Dissociation of nucleotide from noncatalytic sites was illumination dependent. The dissociation kinetics suggested the existence of at least two nucleotide-binding sites with different dissociation rate constants.

Using a fluorescent probe for superoxide, hydroethidine, we have demonstrated that glucose deprivation (GD) activates production of reactive oxygen species (ROS) in cultured cerebellar granule neurons. ROS production was insensitive to the blockade of ionotropic glutamate channels by MK-801 (10 μM) and NBQX (10 μM). Inhibitors of mitochondrial electron transport, i.e. rotenone (complex I), antimycin A (complex III), or sodium azide (complex IV), an inhibitor of mitochondrial ATP synthase—oligomycin, an uncoupler of oxidative phosphorylation—CCCP, a chelator of intracellular Ca2+-BAPTA, an inhibitor of electrogenic mitochondrial Ca2+ transport—ruthenium red, as well as pyruvate significantly decreased neuronal ROS production induced by GD. GD was accompanied by a progressive decrease in the mitochondrial membrane potential and an increase in free cytosolic calcium ions, [Ca2+]i. Pyruvate, BAPTA, and ruthenium red lowered the GD-induced calcium overload, while pyruvate and ruthenium red also prevented mitochondrial membrane potential changes induced by GD. We conclude that GD-induced ROS production in neurons is related to potential-dependent mitochondrial Ca2+ overload. GD-induced mitochondrial Ca2+ overload in neurons in combination with depletion of energy substrates may result in the decrease of the membrane potential in these organelles.

Succinate-dependent Ca2+ accumulation was investigated in rat liver mitochondria and in operation biopsies of patients under states either with prevalence of adrenergic influences (administration of activating doses of adrenaline, acute phase of immobilization stress, initial period after allogenic transplantation, and acute phase of myocardial infarction) or with prevalence of the reciprocal mediator acetylcholine (late period after transplantation, chronic ulcer disease of stomach and duodenum). Adrenaline prevalence leads to increase of succinate-dependent ATP synthesis and Ca2+ accumulation, which is due to known activation of succinate oxidation. However, together with activation, inhibition of these processes was revealed. The inhibition phase prevails under chronic pathology. Therefore, reciprocal regulation of succinate oxidation and succinate-dependent Ca2+ transport in mitochondria occurs in the body in the course of adaptation. The reciprocal regulation of mitochondrial processes is considered as a mechanism of reciprocal regulation of physiological functions.

Cytokines are important regulators of brain function under both normal and pathological conditions. Cytokines can be synthesized by resident cells of the central nervous system (CNS) (vascular endothelium, cells of the blood-brain barrier, parenchymal cells of the CNS) or cells in the lumen of blood vessels, as well as introduced with the bloodstream. The ratio between the quantity of cytokines synthesized in the CNS and those entering it from external sources under various conditions remains poorly understood. In this work, we studied the contribution of mRNAs from non-resident cells to the common pool of cytokine (TNFα, IL-1β, IL-6, IL-10, CX3CL1, and TGFβ1) mRNAs in the rat neocortex, hippocampus, dura matter, pia matter, and choroid plexus. We also evaluated the representation of various populations of resident and non-resident immune cells based on the expression of marker genes (Ncf1, Tbx21, Foxp3, RORγc). The removal of blood by transcardial perfusion led to a decrease in the quantity of the TNFα mRNA in the neocortex and hippocampus and of the IL-1β, IL-6, and IL-10 mRNAs in the dura mater. The mRNA levels of other cytokines in studied structures were not affected by perfusion. Our findings suggest that mRNAs present in the blood can make a significant contribution to the mRNA levels of some cytokines in the CNS; therefore, preliminary perfusion of brain tissue is a necessary stage of experimental design for correct estimation of mRNA content in the brain.

According to one hypothesis, Parkinson’s disease pathogenesis is largely caused by dopamine catabolism that is catalyzed on mitochondrial membranes by monoamine oxidase. Reactive oxygen species are formed as a byproduct of these reactions, which can lead to mitochondrial damage followed by cell degeneration and death. In this study, we investigated the effects of administration of the mitochondrial antioxidant SkQ1 on biochemical, immunohistochemical, and behavioral parameters in a Parkinson-like condition caused by protoxin MPTP injections in C57BL/6 mice. SkQ1 administration increased dopamine quantity and decreased signs of sensory-motor deficiency as well as destruction of dopaminergic neurons in the substantia nigra and ventral tegmental area in mice with the Parkinson-like condition.