Biological Diversity and Remodeling of Cardiolipin in Oxidative Stress and Age-Related Pathologies.

Age-related dysfunctions are accompanied by impairments in the mitochondrial morphology, activity of signaling pathway, and protein interactions. Cardiolipin is one of the most important phospholipids that maintains the curvature of the cristae and facilitates assembly and interaction of complexes and supercomplexes of the mitochondrial respiratory chain. The fatty acid composition of cardiolipin influences the biophysical properties of the membrane and, therefore, is crucial for the mitochondrial bioenergetics. The presence of unsaturated fatty acids in cardiolipin is the reason of its susceptibility to oxidative damage. Damaged cardiolipin undergoes remodeling by phospholipases, acyltransferases, and transacylases, creating a highly specific fatty acyl profile for each tissue. In this review, we discuss the variability of cardiolipin fatty acid composition in various species and different tissues of the same species, both in the norm and at various pathologies (e.g., age-related diseases, oxidative and traumatic stresses, knockouts/knockdowns of enzymes of the cardiolipin synthesis pathway). Progressive pathologies, including age-related ones, are accompanied by cardiolipin depletion and decrease in the efficiency of its remodeling, as well as the activation of an alternative way of pathological remodeling, which causes replacement of cardiolipin fatty acids with polyunsaturated ones (e.g., arachidonic or docosahexaenoic acids). Drugs or special diet can contribute to the partial restoration of the cardiolipin acyl profile to the one rich in fatty acids characteristic of an intact organ or tissue, thereby correcting the consequences of pathological or insufficient cardiolipin remodeling. In this regard, an urgent task of biomedicine is to study the mechanism of action of mitochondria-targeted antioxidants effective in the treatment of age-related pathologies and capable of accumulating not only in vitro, but also in vivo in the cardiolipin-enriched membrane fragments.

Protein Poly(ADP-ribosyl)ation System: Changes in Development and Aging as well as due to Restriction of Cell Proliferation.

It is well known that the number of dividing cells in an organism decreases with age. The average rate of cell division in tissues and organs of a mature organism sharply decreases, which is probably a trigger for accumulation of damage leading to disturbance of genome integrity. This can be a cause for the development of many age-related diseases and appearance of phenotypic and physiological signs of aging. In this connection, the protein poly(ADP-ribosyl)ation system, which is activated in response to appearance of various DNA damage, attracts great interest. This review summarizes and analyzes data on changes in the poly(ADP-ribosyl)ation system during development and aging in vivo and in vitro, and due to restriction of cell proliferation. Special attention is given to methodological aspects of determination of activity of poly(ADP-ribose) polymerases (PARPs). Analysis of relevant publications and our own data has led us to the conclusion that PARP activity upon the addition of free DNA ends (in this review referred to as stimulated PARP activity) is steadily decreasing with age. However, the dynamics of PARP activity measured without additional activation of the enzyme (in this review referred to as unstimulated activity) does not have such a clear trend: in many studies, the presented differences are statistically non-significant, although it is well known that the number of unrepaired DNA lesions steadily increases with aging. Apparently, the cell has additional regulatory systems that limit its own capability of reacting to DNA damage. Special attention is given to the influence of the cell proliferative status on PARP activity. We have systematized and analyzed data on changes in PARP activity during development and aging of an organism, as well as data on differences in the dynamics of this activity in the presence/absence of additional stimulation and on cellular processes that are associated with activation of these enzymes. Moreover, data obtained in different models of cellular aging are compared.

[Estimation of Time-Dependent microRNA Expression Patterns in Brain Tissue, Leukocytes, and Blood Plasma of Rats under Photochemically Induced Focal Cerebral Ischemia].

miRNA expression over different time periods (24 and 48 h) using the quantitative RT-PCR and deep sequencing has been evaluated in a model of photochemically induced thrombosis. A combination of two approaches allowed us to determine the miRNA expression patterns caused by ischemia. Nine miRNAs, including let-7f-5p, miR-221-3p, miR-21-5p, miR-30c-5p, miR-30a-3p, miR-223-3p, miR-23a-3p, miR-22-5p, and miR-99a-5p, were differentially expressed in brain tissue and leukocytes of rats 48 h after onset of ischemia. In addition, six miRNAs were differentially expressed in the brain tissue and blood plasma of rats 24 h after exposure, among which miR-145-3p and miR-375-3p were downregulated and miR-19a-3p, miR-92a-3p, miR-188-5p, and miR-532-5p were upregulated. In our opinion, miR-188-5p and miR-532-5p may be considered to be new potential markers of ischemic injury. The level of miRNA expression tended to increase 48 h after the onset of ischemia in brain tissue and leukocytes, which reflects not only the local response in brain tissue due to inflammation, vascular endothelial dysfunction, and disorders of the permeability of the blood-brain barrier, but also the systemic response of the organism to multifactor molecular processes induced by ischemic injury.

The penetration of 5-oxo-Pro-Arg-Pro into the brain and the major metabolic pathways of this peptide in the rat brain and blood at the intranasal and intravenous administration.

It was shown that the neuroactive peptide 5-oxo-Pro-Arg-Pro (5-oxo-PRP) is detected in the brain in the time interval of 5-120 min after it was intravenously or intranasally administered to rats; the maximum concentration of labeled tripeptide in these modes of administration was observed after 30 and 10 min, respectively. A significant difference in the concentrations of 5-oxo-PRP in the blood and brain (the latter was 50 times lower) during intravenous administration indicates a relatively low permeability of the peptide across the blood-brain barrier. Pharmacokinetic data analysis showed that, when administered intranasally, approximately 45% of the total number of 5-oxo-PRP detectable in the brain in the entire period of study enters via transport from the nasal cavity, and the rest of the peptide enters through the blood-brain barrier from the blood stream. It was found that 5-oxo-PRP in rats is rapidly metabolized forming proteolytic products, mainly amino acids, and degradation products, presumably oxidized these amino acids.

Doxorubicin causes transient activation of protein poly(ADP-ribosyl)ation in H9c2 cardiomyocytes.

Possible involvement of the system of protein poly(ADP-ribosyl)ation in the mechanisms of cardiotoxicity of doxorubicin, one of the most frequently used anticancer drug, was studied in cultures of cardiomyocytes H9c2. The treatment of H9c2 cells with doxorubicin (1 µM) led to a transient (after 6 h of incubation) increase in the nuclear level of poly(ADP-ribosyl)ated proteins. The observed data indirectly indicate the development of genotoxic stress in the doxorubicin-treated cells, probably caused by the stimulatory effects of doxorubicin and its metabolites on the production of reactive oxygen and nitrogen species.

[EFFECT OF PEPTIDE SEMAX ON SYNAPTIC ACTIVITY AND SHORT-TERM PLASTICITY OF GLUTAMATERGIC SYNAPSES OF CO-CULTURED DORSAL ROOT GANGLION AND DORSAL HORN NEURONS].

The influence of long-term culturing (12 days in vitro) of dorsal root ganglion (DRG) and dorsal horn (DH) neurons with peptide Semax on the level of synaptic activity at co-cultures, as well as short-term plasticity in sensory synapses were studied. It has been shown that neuronal culturing with peptide at concentrations of 10 and 100 µM led to increasing the frequency of spontaneous glutamatergic postsynaptic currents in DH neurons to 71.7 ± 1.8% and 93.9 ± 3.1% (n = 6; P < 0.001); Semax has a not significant effect on the amplitude and frequency of miniature glutamatergic currents, but causes an increase of the amplitudes of spontaneous postsynaptic currents, as well as elevates the quantum content. The data show the increase of multivesicular glutamate release efficiency in neural networks of co-cultures following incubation with the peptide. Also Semax (10 and 100 µM) induces changes of the basic parameters of short-term plasticity in sensory synapses: (1) increasing the paired-pulse ratio from 0.53 ± 0.028 (n = 8) to 0.91 ± 0.072 (n = 6, P < 0.01) and 0.95 ± 0.026 (n = 7; P < 0.001); (2) reducing the ratio of the coefficients of variation (CV2/ CV1) from 1.49 ± 0.11 (n = 8) to 1.02 ± 0.09 (n = 6; P < 0.05) and 1.11 ± 0.13 (n = 7; P < 0.0) respectively. The results indicate a stimulating effect of Semax on the activity of glutamatergic synapses in neural networks of co-cultures, as well as the ability of the peptide to effectively modulate the short-term plasticity in sensory synapses.

[The selective toxic effect of dialdehyde derivatives of the pyrimidine nucleosides on human tumor cells].

The impact of a number of synthetic nucleoside derivatives on the growth and survival of cultured human ovarian tumor cells (line SKOV-3) and normal human lung fibroblasts was investigated. It was shown that the dialdehyde derivatives of uridine, 1-ß-D-eritrofuranozyl uracil and 3'-O-ß-D-ribofuranosyl-2'-deoxythymidine, in contrast to their unoxidized counterparts, exert marked toxic effect on SKOV-3 cells. Cultured human fibroblasts were less susceptible to the damaging effect of the dialdehyde nucleosides. The dialdehyde derivative of 1-ß-D-eritrofuranozyl uracil demonstrated greatest differences in the cytotoxic effect on these cultures: inhibition of tumor SKOV-3 cells growth on 50% or more was achieved at the concentrations of this compound ten times lower than in the case of normal fibroblasts.

[Role of angiostatins in diabetic complications].

Angiogenesis is a process through which new blood vessels form from pre-existing vessels. Angiogenesis is regulated by a number of factors of peptide nature. Disbalance of angiogenic system appears to be the major causative factor contributing vascular abnormalities in diabetes mellitus, resulting in various complications. Angiostatins, which are kringle-containing fragments of plasminogen/plasmin, are known to be powerful physiological inhibitors of neovascularization. In the present review, current literature data on peculiarities of production of angiostatins and their functioning at diabetes mellitus are summarized and analyzed for the first time. Also, role of angiostatins in the pathogenesis of typical diabetic complications, including retinopathies, nephropathies and cardiovascular diseases, is discussed. Data presented in this review may be useful for elaboration of novel effective approaches for diagnostics and therapy of vascular abnormalities in diabetes mellitus.

[The Qualitative Analysis of the Amide Derivative of HLDF-6 Peptide and Its Metabolites with the Use of Tritium- and Deuterium-Labeled Derivatives].

The goal of the study was to elaborate the pharmacokinetics methods of the amide derivative of peptide HLDF-6 (TGENHR-NH2) and its range of nootropic and neuroprotective activity is wide. The hexapeptide 41TGENHR46 is a fragment of the HDLF differentiation factor. It forms the basis for the development of preventive and therapeutic preparations for treating cerebrovascular and neurodegenerative conditions. Pharmacokinetic and molecular mechanisms of the action of the HLDF-6 peptide were studied using tritium- and deuterium-labeled derivatives of this peptide, produced with the use of the high-temperature solid-state catalytic isotope exchange reaction (HSCIE). This reaction was employed to produce the tritium-labeled peptide [3H]TGENHR-NH2 with a molar radioactivity of 230 Ci/mmol and the deuterium-labeled peptide [2H]TGENHR-NH2 with an average deuterium incorporation equal to 10.5 atoms. It was shown by the NMR spectroscopy that the isotope label distribution over the labeled peptide's molecule was uniform, which allowed qualitative analysis ofboth the peptide itself and its fragments in the organism's tissues to be conducted. The newly developed pharmacokinetics method makes it possible to avoid almost completely losses of the peptides under study due to biodegradation during the analysis of tissues. These labeled peptides were used in mice, rats and rabbits to study the pharmacokinetics of the peptide and to calculate the values of its principal pharmacokinetic parameters. Characteristics of its pharmacokinetic profile in the blood were obtained, the hypothesis of pharmacokinetics linearity tested, its metabolism analyzed and its bioavailability value, 34%, calculated. It has been shown that the studied TGENHR-NH2 peptide shows high resistance to hydrolysis in the blood plasma, with dipeptidyl aminopeptidases making the largest contribution to its hydrolysis.

[Pretective action of peptide pro-gly-pro on electrophysiological characteristics of cultured hippocampal neurones during excitotoxic damage].

Electrophysiological characteristics of hippocampal neurones cultured with Pro-Gly-Pro peptide were studied using glutamate excitotoxisity model (excitotoxic damage was induced by 100 mkM glutamate application during 5 min). It was found that negative changes in neurones cultured with 10 mcM Pro-Gly-Pro were less prominent if compared with control ones. Culturing with the peptide significantly affected the following parameters: resting potential (-55 +/- 4 mV in control; -29 +/- 6 MV after glutamate application; -38 +/- 5 MV cultured after glutamate application), action potential amplitude (91 +/- 4; 65 +/- 5; 84 +/- 5 mV), duration (4,3 +/- 0,4; 9,5 +/- 1,6; 5,2 +/- 0,7 ms), depolarization (56 [38, 84]; 27 [21, 35]; 46 [28, 62] MV/Ms) and repolarization (-29 [-38, -27]; -20 [-21, -18]; -29 [-33, -22] M(V)/MS) rates. The data obtained suggest that PGP exhibit its neuroprotective properties on a level of basic electrophysiological characteristics, appropriate cellular mechanisms require further investigations.

The protein poly(ADP-ribosyl)ation system: its role in genome stability and lifespan determination.

The processes that lead to violation of genome integrity are known to increase with age. This phenomenon is caused both by increased production of reactive oxygen species and a decline in the efficiency of antioxidant defense system as well as systems maintaining genome stability. Accumulation of different unrepairable genome damage with age may be the cause of many age-related diseases and the development of phenotypic and physiological signs of aging. It is also clear that there is a close connection between the mechanisms of the maintenance of genome stability, on one hand, and the processes of spontaneous tumor formation and lifespan, on the other. In this regard, the system of protein poly(ADP-ribosyl)ation activated in response to a variety of DNA damage seems to be of particular interest. Data accumulated to date suggest it to be a kind of focal point of cellular processes, guiding the path of cell survival or death depending on the degree of DNA damage. This review summarizes and analyzes data on the involvement of poly(ADP-ribosyl)ation in various mechanisms of DNA repair, its interaction with progeria proteins, and the possible role in the development of spontaneous tumors and lifespan determination. Special attention is given to the relationship between various polymorphisms of the human poly(ADP-ribose) polymerase-1 gene and longevity.

[Effect of Mn(II) on the error-prone DNA polymerase iota activity in extracts from human normal and tumor cells].

The DNA polymerase iota (Pol iota), which has some peculiar features and is characterized by an extremely error-prone DNA synthesis, belongs to the group of enzymes preferentially activated by Mn2+ instead of Mg2+. In this work, the effect of Mn2+ on DNA synthesis in cell extracts from a) normal human and murine tissues, b) human tumor (uveal melanoma), and c) cultured human tumor cell lines SKOV-3 and HL-60 was tested. Each group displayed characteristic features of Mn-dependent DNA synthesis. The changes in the Mn-dependent DNA synthesis caused by malignant transformation of normal tissues are described. It was also shown that the error-prone DNA synthesis catalyzed by Pol iota in extracts of all cell types was efficiently suppressed by an RNA aptamer (IKL5) against Pol iota obtained in our work earlier. The obtained results suggest that IKL5 might be used to suppress the enhanced activity of Pol iota in tumor cells.

[Transcriptome alteration in hippocampus under the treatment of tuftsin analog Selank].

The effect of single and course administration of Selank (Thr-Lys-Pro-Arg-Pro-Gly-Pro) in the dose 200 microg/kg on the rat hippocampus transcriptional profile were studied using cDNA microarray technology. It was shown that mRNA levels of 36 genes changed more than 2-fold after a single intranasal Selank administration, and 20 genes--after course administration. It should be noted that most of them encode proteins associated with the plasma membrane (including transmembrane proteins). This suggests that Selank is able to regulate ion homeostasis of hippocampal cells and thereby modulate different ion-dependent processes, which include the processes of learning and memory formation.

Atomic force microscopy study of the arrangement and mechanical properties of astrocytic cytoskeleton in growth medium.

Astrocytes are quite interesting to study because of their role in the development of various neurodegenerative disorders. The present work describes an examination of the arrangement and mechanical properties of cytoskeleton of living astrocytes using atomic force microscopy (AFM). The experiments were performed with an organotypic culture of dorsal root ganglia (DRG) obtained from a chicken embryo. The cells were cultivated on a gelatinous substrate and showed strong adhesion. AFM allows one to observe cytoskeleton fibers, which are interpreted as actin filaments and microtubules. This assumption is supported by confocal microscopy fluorescence imaging of α-tubulin and fibrillar actin. Mapping of the local Young's modulus of a living astrocyte showed that the stiff areas correspond to the sites where the cytoskeleton fibers are located. Thus, the data obtained indicate that AFM is a promising method to study neural cells cytoskeleton integrity and arrangement inin vitromodels of neurodegeneration.

Formation of spatial memory in rats with ischemic lesions to the prefrontal cortex; effects of a synthetic analog of ACTH(4-7).

Photochemically induced thrombosis of blood vessels in the prefrontal cortex in rats was shown to lead to ischemic infarcts in the lesion zone. Bilateral ischemic lesioning of the prefrontal cortex degraded measures of spatial memory when animals were tested in a Morris water maze with an invisible platform 20-24 days after surgery. Chronic intranasal administration of the peptide Met-Glu-His-Phe-Pro-Gly-Pro (Semax), a synthetic analog of ACTH(4-7), at a dose of 250 microg/kg/day during the first six days after photothrombosis, led to recovery of the animals' learning ability. The long-term antiamnestic action of the peptide observed here may result from its neuroprotective activity and its ability to stimulate the synthesis of neurotrophic factors.

[Formation of the spatial memory in rats with ischemic injury in prefrontal areas of the cortex--effects of a sinthetic analogue of ACTH(4-7)].

It was shown, that photochemically induced thrombosis of blood vessels in prefrontal areas of the cortex lead to the formation of well-defined infarct zone. Bilateral prefrontal cortex injury impair acquisition of the Morris water-maze on postoperative days 20-24. Chronic treatment of brain-injuried rats with peptide semax, a synthetic analogue of ACTH(4-7), at a dose of 250 microg/kg (intranasal, once a day) during six days after photothrombosis resulted in restoration of spatial learning ability. The observed long-lasting antiamnesic effects of the semax are probably caused by its strong neuroprotective action, and ability to induce synthesis of nerotrophic factors.

[Effect of semax on the temporary dynamics of brain-derived neurotrophic factor and nerve growth factor gene expression in the rat hippocampus and frontal cortex].

Semax is a synthetic peptide, which consists of the N-terminal adrenocorticotropic hormone fragment (4-7) (ACTH4-7) and C-terminal Pro-Gly-Pro peptide. Semax promotes neuron survival in hypoxia, increases selective attention and memory storage. It was shown that this synthetic peptide exerted a number of gene expressions, especially brain derived neurotrophic factor gene (Bdnf) and nerve growth factor gene (Ngf). Temporary dynamics of Bdnf and Ngf ex- pression in rat hippocampus and frontal cortex under Semax action (50 mg/kg, single intranasal administration) was studied in this work. It was shown that the studied gene expression levels changed significantly both in the hippocampus and the frontal cortex tissues 20 minutes after the peptide preparation application. The expression levels decreased in the hippocampus and increased in the frontal cortex. Forty minutes after Semax administration both gene expression levels returned to the level typical of control tissues. After that they increased significantly by 90 minutes after experiment start. Bdnf and Ngf expression levels decreased up to the control levels by 8 hours after medicine applying maximum gene expression levels were attained. Thus, Semax administration results in rapid, long-term, and specific activation of Bdnf and Ngf expression changes in different rat brain departments.

Neurotrophin gene expression in rat brain under the action of Semax, an analogue of ACTH 4-10.

The heptapeptide Semax, an analogue of the N-terminal adrenocorticotropic hormone fragment (4-10) (ACTH(4-10)), has been shown to exert a number of neuroprotective effects. There are some investigations that connected these effects with the increase of neurotrophin gene expression under the peptide drug application in neuron cell cultures [M.I. Shadrina, O.V. Dolotov, I.A. Grivennikov, P.A. Slominsky, L.A. Andreeva, L.S. Inozemtseva, S.A. Limborska, N.F. Myasoedov, Rapid induction of neurotrophin mRNAs in rat glial cell cultures by Semax, an adrenocorticotropic hormone analogue, Neurosci. Lett. 308 (2001) (2) 115-118]. In this work, we examined the action of Semax on rapid changes of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) gene expression in vivo. Male Wistar rats were treated for 1h with Semax (50 microg/kg, single intranasal application) and neurotrophin gene expression in rat brain was analyzed by real-time polymerase chain reaction (PCR). It was revealed that an intranasal application of Semax increased the expression of both neurotrophin genes in rat hippocampus. Bdnf gene expression also increased in the brainstem and cerebellum. Ngf gene expression decreased in rat frontal cortex. Thus, Semax induces rapid, gene- and region-specific changes in neurotrophin gene expression in normal rat brain.