Biochemistry (Moscow), Supplement Series B: Biomedical Chemistry (v.6, #1)

In-depth scholar literature analysis of Alzheimer’s disease neurodegenerative features of amyloid beta protein neurochemistry modification and excessive phosphorylation of tau protein (and associated neuronal cytoskeleton rearrangements) are secondary phenomena. At early disease stage these neurobiochemical mechanisms are reversible and serve to heal an impairment of biophysical properties of neuronal membranes, neurotransmission, basic neuronal function and neuroplasticity, while preserving anatomical and functional brain fields. Aβ and tau could well serve to biochemically restore physico-chemical properties of neual membranes due to a role these proteins play in lipid metabolism. Under such scenario therapeutic block of aggregation and plaque formation of Aβ and inhibition of tau phosphorylation, as well as pharmaceutical modification of other secondary neurodegenerative features (such as a cascade of oxidative stress reactions) are unable to provide an effective cure of Alzheimer’s disease and related pathologies of the Central and peripheral nervous systems, because they are not arraying primary pathagenetic cause. We review the role of Aβ in compensatory mechanisms of neuroplasticity restoration under normal physiological condition and Alzheimer’s disease.
Keywords: amyloid beta; amyloid plaque; Alzheimer’s disease; hippocampus; cholesterol; phospholipids; synaptic plasticity; lipoproteins

Alzheimer’s disease (AD) is an incurable degenerative disease of the central nervous system, leading to dementia. The basis of AD is a neurodegenerative process that leads to death of neurons in the cerebral cortex. This neurodegenerative process is associated with the formation of neurofibrillary tangles in the brain and the deposition of senile plaques, with beta-amyloid peptide (Aβ) as their main component. The risk factors for AD include age, as well as hypertension, atherosclerosis, diabetes mellitus, and hypercholesterolemia; pathogeneses of all these pathologies involve angiotensin converting enzyme (ACE), the key enzyme of the renin-angiotensin (RAS) and kallikrein-kinin (KKS) systems. Recently it has been found that ACE together with other metallopeptidases, participates in metabolism of Aβ by cleaving peptide bonds at the N-terminal and C-terminal regions of the Aβ molecule. The role of the ACE in the degradation processes of Aβ attracts much interest, which is associated with the fact that prescription of ACE inhibitors is the main therapeutic approach used for treatment of various forms of hypertension and other cardiovascular diseases. However, until now it remains unclear, whether the antihypertensive drugs inhibiting RAS can be used for treatment or prevention of AD. Currently, numerous studies on possible relationship between RAS and AD are carried out.
Keywords: Alzheimer’s disease; beta-amyloid peptide; angiotensin converting enzyme; angiotensin converting enzyme inhibitors

The proteomic study of Danio rerio embryos using one-dimensional electrophoresis and mass-spectrometry by J. S. Kisrieva; N. A. Petushkova; A. S. Chernobrovkin; O. V. Larina; O. P. Trifonova; N. F. Samenkova; G. P. Kuznetsova; I. I. Karuzina; V. N. Kashirtseva; N. F. Belayeva; A. V. Lisitsa (23-30).
Using one-dimensional proteomic mapping (combination of one-dimensional gel electrophoresis (1DE) with subsequent mass spectrometry MALDI-TOF-PMF) the protein profile of Danio rerio embryos has been investigated. The fish species Danio rerio is the most effective alternative model of vertebrates used for studies of drug toxicity (e.g. doxorubicin) due to its high degree of homology with human genome. The proteomic profiling resulted in identification of 84 proteins, including 15 vitellogenins. Using the procedure of preparation of homogenates of Danio rerio embryos optimized by ultrasonic treatment promoting removal of yolk basic proteins (vitellogenin) we have registered changes in the proteome profile of D. rerio embryos induced by doxorubicin (DOX). Growth D. rerio embryos in the medium with DOX caused the decrease in the number of vitellogenins, disappearance of cardiac troponins, and induction of caspase-3. All these observations are consistent with the literature data on doxorubicin-induced cardiotoxicity. The proposed method of 1D proteomic mapping may be used not only for protein identification but also for registration of changes in embryonic proteomic profile caused by drugs or any toxic compound for studying the mechanisms underlying induced toxicity.
Keywords: Danio rerio; embryos; one-dimensional gel electrophoresis; mass spectrometry; protein identification; doxorubicin

Ability of drugs to cross blood-brain barrier (BBB) (BBB+ for BBB-penetrating and BBB- for non-penetrating compounds, respectively) is one of the most important properties of chemicals acting on the central nervous system (CNS). This work presents the results of modelling of the relationship between chemicals structure and BBB-crossing ability. The data set included 1513 compounds BBB+/− (1276 BBB+ and 237 BBB-). Computer modelling of structure-activity relationship was realized by two directions: using the “read-across” method and linear discriminant analysis (LDA) based on physico-chemical descriptors. It was found that a sum of hydrogen bond donor-acceptor factors is the principal parameter, which defines BBB penetration.
Keywords: blood-brain barrier; structural similarity; hydrogen bound; physico-chemical descriptors; HYBOT

Changes of doxorubicin distribution in blood and plasma after its inclusion into nanophospholipid formulations by M. G. Zykova; O. M. Ipatova; V. N. Prozorovskii; N. V. Medvedeva; A. A. Voskresenskaya; T. S. Zakharova; T. I. Torkhovskaya (39-41).
Using original technology developed at the Institute of Biomedical Chemistry (Russian Academy of Medical Sciences) a drug composition based on plant phospholipids and the antitumor drug doxorubicin (particle size <30 nm) has been developed. In vitro experiments demonstrated that doxorubicin inclusion into the phospholipids nanoparticles decreased drug binding to blood cells and thus increase proportion of the potentially active drug in plasma. This was accompanied by changes in drug redistribution from the plasma protein fraction to high density lipoproteins. Importance of these changes for doxorubicin bioavailability and antitumor activity is discussed.
Keywords: doxorubicin; phospholipids nanoparticles; lipoproteins; erythrocytes

Sustained release of the antitumor drug paclitaxel from poly(3-hydroxybutyrate)-based microspheres by A. P. Bonartsev; S. G. Yakovlev; E. V. Filatova; G. M. Soboleva; T. K. Makhina; G. A. Bonartseva; K. V. Shaitan; V. O. Popov; M. P. Kirpichnikov (42-47).
The development of sustained release formulations based on biodegradable polymers is a promising trend in modern pharmacology. Polyhydroxyalkanoates (PHA) attract increasing attention due to their biodegradability and high biocompatibility, which make them suitable for the development of novel drug dosage forms. We have produced poly(3-hydroxybutyrate) (PHB)-based microspheres loaded with the antitumor drug paclitaxel and investigated morphology, drug release kinetics and the effect of these microspheres on tumor cells in vitro. The data on the kinetics of drug release, biocompatibility and biological activity of the biopolymer microspheres in vitro have demonstrated that the studied system of prolonged drug release had lower toxicity and higher efficiency compared to the traditional dosage forms of paclitaxel.
Keywords: poly(3-hydroxybutyrate); paclitaxel; microspheres; sustained release; antitumor

The influence of compound aITEL1296 on telomerase activity and growth of cancer cells by N. A. Kovalenko; D. D. Zhdanov; M. V. Bibikova; V. Y. Gotovtseva (48-54).
Telomerase is a ribonucleoprotein complex, which synthesizes telomeric repeats and which has been identified as a promising target for anticancer therapy. Here we have investigated the effect of a new compound aITEL1296 on telomerase activity. aITEL1296 effectively inhibited telomerase activity; its inhibitory activity was a bit higher (IC50 = 0.19 ± 0.02 ng/mL) than that of BIBR1532, one of the most potent telomerase inhibitors known to date. In addition to telomerase inhibition aITEL1296 activated apoptotic mechanisms and effectively suppressed proliferation of tumor cell lines (GI50 = 5.0 ± 0.2 ng/mL for most sensitive cell line LnCap) but not normal fibroblast cell line.
Keywords: telomerase; telomere; aITEL1296; inhibitor; cancer cells; apoptosis

The role of inhibition of NO formation in the metabolic recovery of ischemic rat heart by apelin-12 by O. I. Pisarenko; Yu. A. Pelogeykina; V. S. Shulzhenko; I. M. Studneva; Zh. D. Bespalova; A. A. Az’muko; M. V. Sidorova; M. E. Pal’keeva (55-60).
The effects of apelin-12, a 12 amino acid peptide (H-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe-OH, A-12), on recovery of energy metabolism and cardiac function have been studied in isolated working rat hearts perfused with Krebs buffer (KB) containing 11 mM glucose and subjected to global ischemia and reperfusion. Infusion of 140 μM A-12 before ischemia enhanced myocardial ATP, the total pool of adenine nucleotides (ΣAN = ATP+ADP+AMP) and the energy charge of cardiomyocytes ((ATP + 0.5ADP)/ΣAN) at the end of reperfusion compared with control (KB infusion) and decreased lactate content and lactate/pyruvate ratio in the reperfused myocardium up to the initial values. This was accompanied by improved recovery of coronary flow and cardiac function. Co-administration of A-12 and 100 μM L-NAME (an inhibitor of NO synthases) significantly attenuated the A-12 effects on metabolic and functional recovery of reperfused hearts. These results indicate involvement of NO in mechanisms of cardioprotection that are tightly associated with recovery of energy metabolism in the postischemic heart.
Keywords: apelin-12; myocardial ischemia and reperfusion; energy metabolism; NO

Thermosensitization of tumor cells with inhibitors of chaperone activity and expression by V. A. Kudryavtsev; Yu. M. Makarova; A. E. Kabakov (61-67).
Effects of inhibitors of the heat shock protein 90 (HSP90) chaperone activity and inhibitors of the heat shock protein (HSP) expression on sensitivity of HeLa tumor cells to hyperthermia were studied. It was found that nanomolar concentrations of inhibitors of the HSP90 activity (17AAG or radicicol) slowed down the chaperone-dependent reactivation of a thermolabile reporter (luciferase) in heat-stressed HeLa cells and slightly enhanced their death following the incubation for 60 min at 43°C. The inhibitors of HSP90 activity stimulated de novo induction of additional chaperones (HSP70 and HSP27) that significantly increased intracellular HSP levels. Treatment of the cells with 17AAG or radicicol along with an inhibitor of the HSP induction (e.g. quercetin or triptolide, or NZ28) completely prevented the increase in the intracellular chaperone levels resulting from the inhibition of HSP90 activity and subsequent heating. Combination of all three treatments (inhibition of the HSP90 activity + inhibition of the HSP induction + heating at 43°C for 60 min) resulted in more potent inhibition of the reporter reactivation and a sharp (2–3-fold) increase in cell death. Such enhancement of the cytotoxicity may be attributed to the “chaperone deficiency” when prior to heat stress both the functional activity of constitutive HSP90 and the expression of additional (inducible) chaperones are blocked in the cells.
Keywords: heat shock proteins; HSF1; thermoresistance; hyperthermia

Fragments of cell-free DNA increase transcription in human mesenchymal stem cells, activate TLR-dependent signal pathway, and suppress apoptosis by S. V. Kostyuk; E. M. Malinovskaya; A. V. Ermakov; T. D. Smirnova; L. V. Kameneva; O. V. Chvartatskaya; P. A. Loseva; E. S. Ershova; L. N. Lyubchenko; N. N. Veiko (68-74).
Human mesenchymal stem cells (MSCs) are widely used in regenerative medicine. However, many questions on the role of different signaling pathways in the regulation of stem cell (SC) functional activity within the organism still remain unanswered. In lesion regions the level of cell death increases and DNA fragments from dead cells (cell-free DNA, cfDNA) are accumulated in blood. We have shown that in vitro contact of adipose-derived MSCs with cfDNA fragments the increase of transcription activity (evaluated by the increase of total cellular RNA and rRNA) is observed. GC-rich cfDNA fragments (GC-DNA) activated the TLR9-dependent signal pathway causing up-regulation of expression of TLR9 and MyD88, the TLR9-signaling pathway adapter. AT-rich DNA fragments did not increase the TLR9 expression, but did increase the level of MyD88 mRNA. So we suggest that AT-DNA acts via some other receptors, which, nevertheless, activate MyD88-dependent signalling in MSCs. We have also shown that cfDNA fragments decreased the activity of caspase, an apoptotic enzyme. Thus, cfDNA can significantly influence the functional activity of MSC by activating the TLR9- and MyD88-dependent signal pathways and by lowering the apoptosis level.
Keywords: mesenchymal stem cells; cell-free DNA; TLR9; apoptosis

Sulfated polysaccharides of brown seaweeds are ligands of toll-like receptors by I. D. Makarenkova; D. Yu. Logunov; A. I. Tukhvatulin; I. B. Semenova; T. N. Zvyagintseva; V. I. Gorbach; S. P. Ermakova; N. N. Besednova (75-80).
The interaction of sulfated polysaccharides (fucoidans) from brown seaweeds Laminaria japonica, Laminaria cichorioides, and Fucus evanescens with Toll-like receptors (TLRs) expressed on membranes of human embryonic kidney epithelial cells (HEK293-null, HEK293-TLR2/CD14, HEK293-hTLR4/CD14-MD2 and HEK293-hTLR2/6), has been investigated. In vitro fucoidans specifically interacted with TLR-2, TLR-4, and the heterodimer TLR-2/6; this resulted in activation of the transcription nuclear factor NF-κB. Composition the hydrolyzed fucoidan from F. evanescens was analyzed by gas-liquid chromatography and chromatography-mass spectrometry. Results indicated the absence of 3-hydroxytetradecanoic acid (3-OHC14), the basic fatty acid component of lipopolysaccharides in the analyzed preparation. Thus, the obtained results suggest that fucoidans from brown seaweeds possessing immunotropic activity are independent ligands for TLRs. They can induce genetically determined biochemical processes of protection organisms against pathogenic microorganisms.
Keywords: toll-like receptors; transcription nuclear factor NF-κB; fucoidan; lipopolysaccharide; gas-liquid chromatography

The effect of natural dicarbonyls on activity of antioxidant enzymes in vitro and in vivo by V. Z. Lankin; G. G. Konovalova; A. K. Tikhaze; L. V. Nedosugova (81-86).
Natural dicarbonyls, which may be accumulated during oxidative stress in atherosclerosis (e.g. malondialdehyde) or carbonyl stress in diabetes mellitus (glyoxal and methylglyoxal) effectively inhibited activities of commercial preparations of the antioxidant enzymes: Cu,Zn-superoxide dismutase (Cu,Zn-SOD) and Se-contained glutathione peroxidase from human and bovine erythrocytes, and also rat liver glutathione-S-transferase. After incubation of human erythrocytes with 10 mM of each investigated dicarbonyls the decrease of intracellular Cu,Zn-SOD was observed. The decreased activity of erythrocyte Cu,Zn-SOD was also detected in patients with diabetes mellitus type 2 with carbohydrate metabolism impairments but effective sugar-lowered therapy was accompanied by the increase of this enzyme activity. The increase of erythrocytes Cu,Zn-SOD activity in diabetic patients treated with metformin (which may utilize methylgly-oxal) was higher than in erythrocytes of diabetic patients subjected to traditional therapy.
Keywords: free radical oxidation; malondialdehyde; glyoxal; methylglyoxal; antioxidant enzymes; Cu,Zn-superoxide dismutase; metformin

The influence of B-group vitamins on monooxygenase activity of cytochrome P450 3A4: Pharmacokinetics and electro analysis of the catalytic properties by V. V. Shumyantseva; E. V. Shich; A. A. Machova; T. V. Bulko; V. G. Kukes; O. S. Sizova; G. V. Ramenskaya; S. A. Usanov; A. I. Archakov (87-93).
Simultaneous administration of loading doses of B-group vitamins and diclofenac allow to decrease the daily dose of this drug without reduction of its analgesic effect. In all three schemes of the diclofenac intake (diclofenac alone, diclofenac plus 2 tablets of Gitagamp (a mixture of B-group vitamins), and diclofenac plus 4 tablets of Gitagamp—maximal concentration of blood diclofenal (Cmax) was observed 1 h after the treatment. In the case of diclofenac treatment alone, with 2 tablets of Gitagamp, and with 4 tablets of Gitagamp Cmax values were 1137.2 ± 82.4, 1326.7 ± 122.5 and 2200.4 ± 111.3 ng/mL, respectively. Thus, loading doses of B-group vitamins caused a statistically significant effect on the Cmax value of blood diclofenac concentration; they also reduced manifestations of the pain syndrome. Pharmacodynamics and pharmacokinetics data were confirmed in electrochemical studies of cytochrome P450 3A4 (CYP3A4) activity. This enzyme was immobilized onto screen printed graphite electrodes modified with gold nanoparticles and synthetic membrane-like compound didodecyldimethylammonium bromide (DDAB/Au). Electrochemical analysis revealed the influence of B-group vitamins on metabolism of the non-steroidal anti-inflammatory drug diclofenac catalyzed by cytochrome P450 3A4. Comparative analysis of the effect of 300 μM vitamins of the B-group (B1, B2, and B6) demonstrated that riboflavin was the most effective inhibitor of diclofenac hydroxylation catalyzed by CYP3A4. These data support possibility of regulation of pharmacokinetic parameters and manifestation of pharmacodynamic effects by loading doses of B-group vitamins, which regulate the catalytic activity of drug metabolizing enzymes such as cytochrome P450 3A4.
Keywords: cytochrome P450 3A4; pharmacokinetics; diclofenac; B-group vitamins; electrochemistry; enzyme electrodes

Kinetic and thermodynamic analysis of dimerization inhibitors binding to HIV protease monomers by surface plasmon resonance by P. V. Ershov; O. V. Gnedenko; A. A. Molnar; A. V. Lisitsa; A. S. Ivanov; A. I. Archakov (94-97).
The analysis of kinetic and thermodynamic parameters of binding of peptide and nonpeptide dimerization inhibitors of HIV protease (HIVp) to the enzyme monomers immobilized on an optical chip has been studied by surface plasmon resonance. The molecular interactions were investigated at different inhibitor concentrations (0–80 μM) and temperatures (15–35°C). Determination of kinetic (k on, k off), equilibrium (K d), and thermodynamic (ΔG, ΔH, and -TΔS) has shown that both inhibitors are characterized by similar interaction parameters and the entropic term (-TΔS) of about −20 kcal/mol is the main driving force for the HIVp complex formation with the inhibitors, while the positive value (14 kcal/mol) of the enthalpic term (ΔH) counteracted the complex formation.
Keywords: HIV-1 protease; optical biosensor; dimerization inhibitor; surface plasmon resonance; thermodynamics