Biochemistry (Moscow) (v.81, #5)

Effect of fibrinogen on platelet reactivity measured by the VerifyNow P2Y12 assay by A. B. Dobrovolsky; P. S. Laguta; E. V. Guskova; E. B. Yarovaya; E. V. Titaeva; A. N. Storozhilova; E. P. Panchenko (439-444).
The VerifyNow assay is based upon the ability of activated platelets to cross-link beads coated with fibrinogen. However, fibrinogen is an abundant protein of blood, and therefore it may affect test results by competing with fibrinogen of beads for binding to platelets. To test this assumption, we assessed the influence of artificial alteration of fibrinogen level in blood samples obtained from donors (n = 9) and patients on clopidogrel therapy (n = 8) on the results of the VerifyNow P2Y12 assay. Fibrinogen level was altered by adding to blood samples 1/10 volume of fibrinogen solution (10.56 g/liter) or corresponding buffer. Relative to baseline, addition of buffer significantly increased platelet reactivity, whereas addition of fibrinogen decreased it. Analysis of the relationship between change in platelet reactivity values (dBase and dPRU) and change in fibrinogen concentration (dFg) revealed strong negative correlations: dBase =–63.3 × dFg–27.1 (r =–0.924, p < 0.0005) and dPRU =–54.4 × dFg–21.8 (r =–0.764, p < 0.0005). Thus, the results of our experiments suggest that: (i) blood fibrinogen strongly influences results of the VerifyNow P2Y12 assay, and (ii) correcting for fibrinogen effect may be needed to improve the accuracy of the test in the measuring of antiplatelet effect of clopidogrel therapy.
Keywords: platelets; fibrinogen; P2Y12 inhibitors; VerifyNow test

Apoptosis in cryopreserved eukaryotic cells by M. A. Savitskaya; G. E. Onishchenko (445-452).
This review considers apoptosis mechanisms that have been revealed in cryopreserved cells and which can be controlled using different chemical agents, thereby improving the viability of cells after their return to normal conditions. The role of oxidative stress as of the most significant damaging factor is discussed, as well as the reasonability of including antioxidants into cryopreservation/thawing protocols as independent agents or in combination with other compounds.
Keywords: apoptosis; cryopreservation; oxidative stress; autophagy

Biogenesis of micronuclei by O. P. Kisurina-Evgenieva; O. I. Sutiagina; G. E. Onishchenko (453-464).
The presence of micronuclei in a cell is an indicator of DNA damage and genetic instability. In this review, mechanisms of emergence of micronuclei, their functional activity, and pathways of elimination are discussed. It is supposed that morphological and functional varieties of micronuclei as well as their degradation pathways can be determined by the chromosomal material localized inside these cell structures.
Keywords: micronucleus; replication; transcription; repair; elimination

Superoxide dismutases (SODs) are key enzymes functioning as the first line of antioxidant defense by virtue of the ability to convert highly reactive superoxide radicals to hydrogen peroxide and molecular oxygen. SOD plays a central role in protecting plants against the toxic effects of reactive oxygen species generated during normal cellular metabolic activity or as a result of various environmental stresses. Our review focuses on the characteristics of expression of SOD genes, the mechanisms regulating expression of SOD genes at transcriptional, posttranscriptional, and translation levels, and their functional role(s) during development and in response to biotic or abiotic stresses. We propose two important research directions: studying SOD at the genome-wide or proteome-wide level, and improving plant stress tolerances by selecting varieties using transgenic technology.
Keywords: superoxide dismutase; reactive oxygen species; plants; stress tolerance; expression and regulation

Bacterial sodium-dependent decarboxylases were the first enzymes exemplifying sodium-dependent bioenergetics. These enzyme complexes couple decarboxylation of organic acids with the export of sodium ions via a special membrane subunit. In 711 representative prokaryotic genomes, we have analyzed genomic neighborhoods of the genes that code the membrane subunit of sodium decarboxylases. In representatives of Thermotogae, the operons with the gene of this subunit lack the genes of subunits that perform non-oxidative decarboxylation. Instead, these operons contain the genes of alphaand delta-subunits of decarboxylating oxidoreductases of alpha-ketoacids. The genes of betaand gamma-subunits of the decarboxylating oxidoreductases were found within the genomes of respective Thermotogae species as separate, twogene operons. We suggest that the described two operons code together for sodium-translocating decarboxylating oxidoreductases capable of coupling oxidative decarboxylation of alpha-ketoacids with the export of sodium ions, which is a novel type of bioenergetic coupling.
Keywords: sodium transport; membrane bioenergetics; comparative genomics; phylogenetic analysis; ferredoxin; molecular evolution; anaerobic bacteria

Role of vascular endothelial growth factor (VEGF) in thymus of mice under normal conditions and with tumor growth by E. P. Kisseleva; A. V. Krylov; I. V. Lyamina; I. V. Kudryavtsev; V. I. Lioudyno (491-501).
In our study, we for the first time investigated a role for VEGF as a factor regulating transendothelial migration of murine thymocytes in vitro. Effects of VEGF were examined in a model of thymocyte migration across a monolayer of EA.hy 926 endothelial cells. We showed that VEGF enhanced transendothelial migration of murine thymocytes and their adhesion to endothelial cells in a dose-dependent manner. VEGF did not influence thymocytes, but rather acted on endothelial cells by upregulating surface expression of adhesion molecule ICAM-1 and downregulating activity of 5′nucleotidase. Effects from VEGF were comparable with those from TNF-α. Because it is known that administration of VEGF to intact animals results in thymic atrophy, it was assumed that it might play a role in developing thymic involution during tumor growth. Enhanced egress of thymocytes to the periphery was considered as a plausible mechanism underlying effects of VEGF. However, we revealed no difference in parameters of in vitro transendothelial migration for thymocytes from animals bearing a transplantable hepatoma 22a compared to control animals. VEGF mRNA expression in lysates of thymic stroma was found to be upregulated in mice with grafted tumors, whereas at the protein level the amount of VEGF did not differ. While examining expression of VEGF receptors on thymocytes by flow cytometry, both VEGFR-1 and VEGFR-2 were not detected, whereas the percentage of Nrp-1-positive thymocytes in animals with hepatoma 22a was as high as in the control group. Thus, we were unable to confirm a hypothesis regarding participation of VEGF in developing thymic involution during progression of experimental hepatoma. However, a set of novel data concerning a role for VEGF in stimulating transendothelial migration of thymocytes in vitro was obtained, and it may be of significance for understanding mechanisms underlying thymus functioning as well as a role of this cytokine in preparing endothelial cells for egress of thymocytes to the periphery.
Keywords: thymus; vascular endothelial growth factor (VEGF); migration; adhesion; endothelial cells; VEGF receptors; activity of 5′-nucleotidase

Staphylococcus simulans recombinant lysostaphin: Production, purification, and determination of antistaphylococcal activity by I. S. Boksha; N. V. Lavrova; A. V. Grishin; A. V. Demidenko; A. M. Lyashchuk; Z. M. Galushkina; R. S. Ovchinnikov; A. M. Umyarov; L. R. Avetisian; M. Iu. Chernukha; I. A. Shaginian; V. G. Lunin; A. S. Karyagina (502-510).
Staphylococcus simulans lysostaphin is an endopeptidase lysing staphylococcus cell walls by cleaving pentaglycine cross-bridges in their peptidoglycan. A synthetic gene encoding S. simulans lysostaphin was cloned in Escherichia coli cells, and producer strains were designed. The level of produced biologically active lysostaphin comprised 6-30% of total E. coli cell protein (depending on E. coli M15 or BL21 producer) under batch cultivation conditions. New methods were developed for purification of lysostaphin without affinity domains and for testing its enzymatic activity. As judged by PAGE, the purified recombinant lysostaphin is of >97% purity. The produced lysostaphin lysed cells of Staphylococcus aureus and Staphylococcus haemolyticus clinical isolates. In vitro activity and general biochemical properties of purified recombinant lysostaphin produced by M15 or BL21 E. coli strains were identical to those of recombinant lysostaphin supplied by SigmaAldrich (USA) and used as reference in other known studies. The prepared recombinant lysostaphin represents a potential product for development of enzymatic preparation for medicine and veterinary due to the simple purification scheme enabling production of the enzyme of high purity and antistaphylococcal activity.
Keywords: Staphylococcus simulans ; Escherichia coli ; recombinant lysostaphin; heterologous expression; liquid chromatography; lytic activity; Staphylococcus sp. clinical isolates

Neuroprotective effect of carnosine on primary culture of rat cerebellar cells under oxidative stress by A. V. Lopachev; O. M. Lopacheva; D. A. Abaimov; O. V. Koroleva; E. A. Vladychenskaya; A. A. Erukhimovich; T. N. Fedorova (511-520).
Dipeptide carnosine (β-alanyl-L-histidine) is a natural antioxidant, but its protective effect under oxidative stress induced by neurotoxins is studied insufficiently. In this work, we show the neuroprotective effect of carnosine in primary cultures of rat cerebellar cells under oxidative stress induced by 1 mM 2,2′-azobis(2-amidinopropane)dihydrochloride (AAPH), which directly generates free radicals both in the medium and in the cells, and 20 nM rotenone, which increases the amount of intracellular reactive oxygen species (ROS). In both models, adding 2 mM carnosine to the incubation medium decreased cell death calculated using fluorescence microscopy and enhanced cell viability estimated by the MTT assay. The antioxidant effect of carnosine inside cultured cells was demonstrated using the fluorescence probe dichlorofluorescein. Carnosine reduced by half the increase in the number of ROS in neurons induced by 20 nM rotenone. Using iron-induced chemiluminescence, we showed that preincubation of primary neuronal cultures with 2 mM carnosine prevents the decrease in endogenous antioxidant potential of cells induced by 1 mM AAPH and 20 nM rotenone. Using liquid chromatographymass spectrometry, we showed that a 10-min incubation of neuronal cultures with 2 mM carnosine leads to a 14.5-fold increase in carnosine content in cell lysates. Thus, carnosine is able to penetrate neurons and exerts an antioxidant effect. Western blot analysis revealed the presence of the peptide transporter PEPT2 in rat cerebellar cells, which suggests the possibility of carnosine transport into the cells. At the same time, Western blot analysis showed no carnosine-induced changes in the level of apoptosis regulating proteins of the Bcl-2 family and in the phosphorylation of MAP kinases, which suggests that carnosine could have minimal or no side effects on proliferation and apoptosis control systems in normal cells.
Keywords: carnosine; AAPH; rotenone; oxidative stress; neuron; antioxidant; neuroprotective effect

Besides regular cap-dependent translation of mRNA, eukaryotes exploit internal initiation of translation driven by internal ribosome entry sites (IRESs). It is supposed that internal initiation provides translation of cellular mRNAs under stress conditions where the cap-dependent initiation is reduced. A number of IRESs have been characterized in mammalian mRNAs, but only a few examples are known in lower eukaryotes, particularly in yeasts. Here we identified two IRESs in the thermotolerant methylotrophic yeast Hansenula polymorpha DL-1. These sites are located in 5′-untranslated regions of genes HPODL_02249 and HPODL_04025 encoding a hypothetical membrane protein and actin-binding protein, respectively. In Saccharomyces cerevisiae cells, both IRESs drive expression of a second gene of a bicistronic mRNA, as well as translation of hairpin-containing monocistronic mRNA. The possibility of spurious splicing or presence of a cryptic promoter in the IRES sequences was ruled out, indicating that expression of a second gene of a bicistronic mRNA was IRESdependent. We evaluated IRES activity of both elements and found that under normal physiological conditions its contribution to the overall translation of the respective mRNAs in yeast cells is about 0.3-0.4%. Therefore, these results suggest that the IRES-dependent translation initiation mechanism exists in Hansenula polymorpha.
Keywords: mRNA; translation; internal initiation of translation; IRES; yeast; Hansenula polymorpha DL-1

Isolation of homogeneous polysaccharide monooxygenases from fungal sources and investigation of their synergism with cellulases when acting on cellulose by A. G. Bulakhov; A. V. Gusakov; A. V. Chekushina; A. D. Satrutdinov; A. V. Koshelev; V. Yu. Matys; A. P. Sinitsyn (530-537).
Lytic polysaccharide monooxygenases (PMO) discovered several years ago are enzymes classified as oxidoreductases. In nature, they participate in microbial degradation of cellulose together with cellulases that belong to the hydrolytic type of enzymes (class of hydrolases). Three PMO from ascomycetes–Thielavia terrestris, Trichoderma reesei, and Myceliophthora thermophila–were isolated and purified to homogeneous state using various types of chromatography. The first two enzymes are recombinant proteins heterologously expressed by the Penicillium verruculosum fungus, while the third is a native PMO secreted by M. thermophila. When acting on microcrystalline cellulose, all these PMOs displayed synergism with the cellulase complex of the P. verruculosum fungus. Replacing 10% of cellulases (by protein concentration) with PMO in the presence of 6.25 mM gallic acid or 2.5 μM of cellobiose dehydrogenase from M. thermophila, used as electron donors for PMO, resulted in the 17-31% increase in the yield of reducing sugars after 24-48 h of the enzymatic reaction.
Keywords: cellulose biodegradation; ascomycetes; polysaccharide monooxygenase; cellulase complex; synergism

Determination of size of folding nuclei of fibrils formed from recombinant Aβ(1-40) peptide by E. I. Grigorashvili; O. M. Selivanova; N. V. Dovidchenko; U. F. Dzhus; A. O. Mikhailina; M. Yu. Suvorina; V. V. Marchenkov; A. K. Surin; O. V. Galzitskaya (538-547).
We have developed a highly efficient method for purification of the recombinant product Aβ(1-40) peptide. The concentration dependence of amyloid formation by recombinant Aβ(1-40) peptide was studied using fluorescence spectroscopy and electron microscopy. We found that the process of amyloid formation is preceded by lag time, which indicates that the process is nucleation-dependent. Further exponential growth of amyloid fibrils is followed by branching scenarios. Based on the experimental data on the concentration dependence, the sizes of the folding nuclei of fibrils were calculated. It turned out that the size of the primary nucleus is one “monomer” and the size of the secondary nucleus is zero. This means that the nucleus for new aggregates can be a surface of the fibrils themselves. Using electron microscopy, we have demonstrated that fibrils of these peptides are formed by the association of rounded ring structures.
Keywords: amyloid fibril; protofibril; oligomer; seed; Aβ peptide; Alzheimer’s disease; electron microscopy