Biochemistry (Moscow)最新文献

One of the fundamental problems in studying addiction is elucidation of mechanisms of alcohol dependence (AD) development. Disturbances of cellular redox balance and inflammation play an important role in AD pathogenesis. Deciphering associations between biological and clinical indicators can elucidate molecular mechanisms of disease pathogenesis. The aim of the work was to study peripheral markers of oxidative stress in patients with AD during early abstinence period and to identify their relationship with clinical parameters of the disease and inflammatory factors. In total, 84 patients with AD were studied (average age, 44.3 ± 8.2 years). The analyzed clinical parameters included patient's age, age of alcohol withdrawal syndrome (AWS) formation, disease duration, and AWS duration. The markers of oxidative stress determined in the blood plasma were oxidation products of proteins (protein carbonyls, PCs), lipids (thiobarbituric acid-reactive products, TBA-RPs), and DNA (8-hydroxy-2'-deoxyguanosine, 8-OH-dG). The content of inflammatory mediators, such as proinflammatory cytokines (IFNγ, IL-1β, IL-6, IL-8, IL-17A, TNFα) was determined in the blood serum. Blood samples of 80 conditionally healthy men (average age, 40.9 ± 9.6 years) were used as a control. Patients with AD demonstrated an increase in the content of PCs, TBA-RPs, and all analyzed cytokines (but not 8-OH-dG) compared to the control individuals. There was a direct correlation between the TBA-RP content and disease duration and inverse correlation of the PC content with the age of AWS formation and AWS duration. The content of PCs demonstrated an inverse correlation with the IL-6 concentration in the blood plasma. We also observed a positive correlation between 8-OH-dG and IL-6, TBA-RPs and IL-8, and TBA-RPs and TNFα. Therefore, the early abstinence period in patients with AD was characterized by a pronounced oxidative stress and inflammation. The obtained results expand the knowledge on the integrative contribution of oxidative stress and inflammatory factors to the AD pathogenesis and can be used in the development of new therapies.

The C3f peptide is a by-product of regulation of the activated complement system with no firmly established function of its own. We have previously shown that C3f exhibits moderate antimicrobial activity against some Gram-positive bacteria in vitro. Presence of two histidine residues in the amino acid sequence of the peptide suggests enhancement of its antimicrobial activity at lower pH and in the presence of metal cations, particularly zinc cations. Since such conditions could be realized in inflammatory foci, the study of dependence of C3f activity on pH and presence of metal cations could provide an opportunity to assess biological significance of antimicrobial properties of the peptide. The peptide C3f and its analogs with histidine residues substituted by lysines or serines, C3f[H/K] and C3f[H/S], were prepared by solid-phase synthesis. Using CD spectroscopy, we found that C3f contained a β-hairpin and unstructured regions; presence of Zn²⁺ did not affect conformation of the peptide. In the present work, it was shown that C3f could also exhibit antimicrobial activity against Gram-negative bacteria, in particular, Pseudomonas aeruginosa ATCC 27583. Exposure of P. aeruginosa and Listeria monocytogenes EGD to the peptide was accompanied by disruption of the barrier function of bacterial membranes. Zn²⁺ ions, unlike Cu²⁺ ions, enhanced antimicrobial activity of C3f against L. monocytogenes, with 4- and 8-fold molar excess of Zn²⁺ being no more effective than a 20% excess. Activity of the C3f analogs was also enhanced to some extent by the zinc ions. Thus, we hypothesize existence of the histidine-independent formation of C3f-Zn²⁺ complexes leading to increase in the total charge and antimicrobial activity of the peptide. In the presence of 0.15 M NaCl, C3f lost its antimicrobial activity regardless of the presence of Zn²⁺, indicating an insignificant role of C3f as an endogenous antimicrobial peptide. Presence of C3f eliminated bactericidal effect of Zn²⁺ against the zinc-sensitive Escherichia coli strain ESBL 521/17, indirectly confirming interaction of the peptide with Zn²⁺. Activity of C3f against Micrococcus luteus A270 increased with decreasing pH, while effect of pH on the C3f activity against L. monocytogenesis was more complex. In this work, we show significance of the factors such as pH and metal cations in realization of antimicrobial activity of peptides based on the example of C3f.

Quite often, conception of a child occurs after consuming small doses of alcohol. However, effect of this factor on offspring has not been studied at all. The aim of this study was to examine the level of motor activity, anxiety-like and depressive-like behavior, sensitivity to analgesic effect of ethanol, as well as activity of the enzymes DPP-IV, PEP, and ADG in the blood of rats whose fathers received ethanol immediately before mating. As a result of the conducted experiments, it was found that the males conceived by the intoxicated fathers have significant differences in behavior compared to control animals. Thus, motor activity in the rats conceived by males under the influence of alcohol was 2-2.5 times less intense; they exhibited decreased severity of the anxiety-like and depressive-like behavior. In such animals, activity of DPP-IV and ADG was increased and activity of PEP in the blood was reduced. In the rats conceived by the fathers under the influence of alcohol, analgesic effect of ethanol was decreased, and there was also reduction in response of the activities of ADG, DPP-IV, and PEP enzymes to ethanol administration. It is assumed that a single use of ethanol by male rats immediately before mating leads to the decrease in methylation of the paternal inherited genes in offspring. As a result, activity of a number of enzymes could change, which leads to the change in the balance of neuropeptides involved in mediation of animal behavior.

Substance use disorders are associated with structural and functional changes in the neuroendocrine, neuromediator, and neuromodulator systems in brain areas involved in the reward and stress response circuits. Chronic intoxication provokes emergence of somatic diseases and aggravates existing pathologies. Substance use disorders and somatic diseases often exacerbate the clinical courses of each other. Elucidation of biochemical pathways common for comorbidities may serve as a basis for the development of new effective pharmacotherapy agents, as well as drug repurposing. Here, we discussed molecular mechanisms underlying integration of visceral systems into the central mechanisms of drug dependence.

The accumulated evidence suggests that varying levels of tyrosine kinase receptor signaling pathway activity may regulate opiate-associated neuroadaptation of noradrenergic system. Neurotrophin-3 (NT-3) interacts with tropomyosin receptor kinases (TRKs), binding mainly to TRKC receptors, which are expressed within noradrenergic neurons in the blue spot (locus coeruleus, LC). Considering the difficulties in delivering full-length neurotrophins to the CNS after systemic administration, low-molecular mimetics of loop 4 in NT-3, hexamethylenediamide bis-(N-monosuccinyl-L-asparaginyl-L-asparagine) (GTS-301), and hexamethylenediamide bis-(N-γ-oxybutyryl-L-glutamyl-L-asparagine) (GTS-302), activating TRKC and TRKB receptors, were synthesized. The aim of the study is comparative examination of the effects of NT-3 dipeptide mimetics on the signs of morphine withdrawal in outbred white rats with opiate dependence, as well as investigation of activation of postreceptor signaling pathways by the mimetics. Dipeptides GTS-301 and GTS-302 after acute administration at doses of 0.1, 1.0, and 10.0 mg/kg (i.p., intraperitoneal) had a dose-dependent effect on the specific morphine withdrawal symptoms with the most effective dose being 1.0 mg/kg. Maximum decrease in the total index of morphine withdrawal syndrome for GTS-301 was 31.3% and for GTS-302 - 41.4%. Unlike GTS-301, GTS-302 weakened mechanical allodynia induced by morphine withdrawal, reducing tactile sensitivity. When studying activation of the postreceptor signaling pathways by the NT-3 mimetics in the HT-22 hippocampal cell culture, a different pattern of postreceptor signaling was shown: GTS-302 (10^-6 M), similar to NT-3, activates all three MAPK/ERK, PI3K/AKT/mTOR, and PLCγ1 pathways, while GTS-301 (10^-6 M) triggers only MAPK/ERK and PLCγ1 pathways. Thus, the identified features of attenuation of the morphine withdrawal syndrome in the rats under GTS-301 and GTS-302 effects could be associated with different activation pattern of the postreceptor pathways.

Neuroinflammation can be an important factor of many disorders in central nervous system (CNS) including cognitive dysfunction, affective disorders, and addictive behavior associated with prenatal alcohol exposure and presented in early adulthood. In this study we used an experimental rodent model of prenatal alcohol (PA) exposure (consumption of a 10% ethanol solution by female Wistar rats throughout pregnancy), multiplex immunofluorescence analysis of interleukins (IL-1α, IL-1β, IL-3, IL-6, IL-9, and IL-12), tumor necrosis factor (TNF-α), and chemokine CCL5, as well as quantitative real-time PCR to assess the level of cytokine mRNAs in the prefrontal cortex of the sexually mature (PND60) offspring - male and female rats with prenatal alcohol intoxication and control animals. Significant decrease in the content of TNF-α and interleukins IL-1β, IL-3, IL-6, IL-9 was detected in the prefrontal cortex of male, but not in the female PA offspring. Importantly, PA males also showed decrease in the level of TNF-α mRNA in the prefrontal cortex by 45% compared to the control males, which may underlie the detected decrease in its content. Taken together, our study demonstrates that a number of neuroimmune factors are regulated in a sex-specific manner in the prefrontal cortex and are differentially affected in males and females by the prenatal exposure to alcohol. Sex factor must be taken into account when conducting further translational studies of the fetal alcohol spectrum disorders and developing new methods for prevention and therapy.

To design a safe cellular system for testing inhibitors targeting the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2, a genetic construct was engineered containing viral cDNA with two blocks of reporter genes while the genes encoding structural S, E, and M proteins were absent. The first reporter block, consisting of Renilla luciferase and green fluorescent protein (Rluc-GFP), was located upstream of the SARS-CoV-2 5'-UTR. Meanwhile, the second block represented by firefly luciferase and red fluorescent protein (Fluc-RFP) was positioned downstream of the transcription regulatory sequence (TRS-N). While the first block of reporter genes can be transcribed by both viral RdRp and cellular polymerases, the second block can only be transcribed by the viral polymerase according to the Coronaviridae discontinuous transcription mechanism. This allowed us to accurately assess effectiveness of the viral RdRp inhibition. To facilitate the search for nucleoside RdRp inhibitors the cell line was obtained expressing herpes simplex virus thymidine kinase, which provides the first stage of nucleoside phosphorylation. When screening the ability of a number of compounds to inhibit catalytic activity of the SARS-CoV-2 RdRp, we discovered antiviral activity of 2'-amino-2'-deoxyadenosine and adenosine-N1-oxide, which exceeded activity of molnupiravir, a therapeutic agent used in the treatment of COVID-19.

Previous studies have shown that the combined effect of fetal hypoxia and maternal stress hormones predetermines tendency to nicotine addiction in adulthood. This study in rats aimed to investigate the effect of prenatal severe hypoxia (PSH) on acetylcholine metabolism in the developing brain, as well as on expression of acetylcholine receptors chrna4 and chrna7 in both the developing brain and adult brain structures following nicotine consumption. In the developing brain of PSH rats, no changes were found in the activity of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) or disturbances in the acetylcholine levels. However, decreased chrna4 expression was detected on the day 15 of pregnancy, while elevation in the chrna7 expression was observed on the days 15 and 16 of embryogenesis. In adulthood, the consequences of PSH were manifested as decreased expression of chrna4 in the medial prefrontal cortex (PFC), nucleus accumbens (NAacc), and hypothalamus (HT), decreased expression of chrna7 in the PFC and hippocampus (HPC). Whereas, nicotine consumption did not decrease the expression levels of chrna4 and chrna7 compared to the control group in the adult PSH rats. Thus, prenatal hypoxia predisposes to impaired expression of the chrna4 and chrna7 genes in adult rats without affecting acetylcholine metabolism during embryonic development.

Genes of the small nucleolar RNA host gene (SNHG) family may participate in oncogenesis through the regulatory functions of encoded long non-coding RNAs (lncRNAs) and by influencing formation of small nucleolar RNAs and ribosome biogenesis. The aim of this work was to evaluate changes in the methylation levels and extent of co-methylation of the SNHG family lncRNA genes (SNHG1, GAS5/SNHG2, SNHG6, SNHG12, SNHG17) in clinical samples of ovarian cancer (OC) as an indication for the similarity of their roles in oncogenesis. Analysis of a representative set of 122 OC samples by quantitative methylation-specific PCR showed a statistically significant (p < 0.01-0.0001) increase in the methylation level of all five studied lncRNA genes. There was also a correlation between the increased methylation levels of GAS5, SNHG6, and SNHG12 and OC progression (clinical stage, tumor size, and metastasis), indicating possible functional significance of hypermethylation of these genes. For four genes (SNHG1, GAS5, SNHG6, and SNHG12), a statistically significant pairwise positive correlation of methylation levels (co-methylation) was observed (r_s > 0.35; p ≤ 0.001), which was in agreement with the GEPIA 2.0 data (426 OC samples) showing co-expression of these genes (r_s > 0.5; p < 0.001). The correlation between the expression levels of GAS5 and SNHG6 was confirmed by RT-qPCR (r_s = 0.46; p = 0.007). Bioinformatics analysis predicted miRNAs common for the SNHG1, GAS5, SNHG6, and SNHG12 lncRNA and potentially capable of interacting with one or more of these lncRNAs via competing endogenous RNA mechanism, as well as mRNAs, whose expression might be affected by the studied lncRNAs. We also investigated a possible involvement of genes for these mRNAs in oncogenesis-related processes, such as RNA processing and splicing and epithelial-mesenchymal transition. As a result of this work, four SNHG family lncRNAs with coregulation and joint putative biological functions in the pathogenesis of OC were identified.

Dependence on psychoactive substances is a phenomenon that is based on the alterations of common molecular and cellular mechanisms, structures and neuronal networks underlying normal brain functioning and realizing stress response, reinforcement and aversion, learning and memory. As a result, aberrant neuroplasticity states associated with somatic changes are formed, which determine the pathogenesis and symptoms of dependence and at the same time can be considered as targets for the development of therapies for such addictions. An integrative scheme of stress and neuroplastic changes participation in the formation of the vicious circle of substance use disorders based on a holistic approach is presented. This special issue of the journal focuses on the molecular mechanisms of psychoactive substance use disorders.