Morpho-functional state of biomembranes after treatment with alkyl derivatives of 2,4-dinitrophenol

The morphofunctional state of the rat liver biomembranes is studied under conditions of the peroral effect of alkyl dinitrophenols: dinitroorthocresole and secondary 2,4-dinitro-6-butylphenol. It is established that free radicals and products of peroxide lipid oxidation (diene conjugates, hydroperoxides, final products reacting with 2-thiobarbituric acid) are very important for realization of the membrane-tropic action of the above compounds. Possible mechanisms of development of the membrane-damaging action of these xenobiotics and their effect on the state of the mitochondrial respiratory chain are discussed. The membrane-tropic effect of alkyl dinitrophenols is confirmed by the results of electron-microscopic studies.     

Influence of beta-radiation on human erythrocyte surface potential under eicosanoid metabolism inhibition

The aim of the research consisted in the study of influence of beta-radiation on response of erythrocyte surface potential to inhibitors of eicosanoid metabolism enzymes (cyclo-, lipoxygenase and phospholipase A2). It was shown, that inhibitors of phospholipase A2 (quinacrine, 10-100 microM), cyclooxygenase (aspirin, 10-100 microM) and cyclo- and lipoxygenase (BW755c, 1-100 microM) lowered electrophoretic mobility (EPM) of erythrocytes by 20-30%. An analogous effect can be exerted by beta-radiation. Nonradioactive leucine in the studied concentrations cannot simulate EPM erythrocytes. Response of cellular EPM to these inhibitors depended on their concentration in the incubation medium. Addition of 14C to the incubation medium changed response of EPM of cells to inhibitors of cyclo- and lipoxygenase but not to quinacrine. However beta-radiation fully abolished the stimulative action of nonspecific activator of phospholipase A2 (Ca-independent), H2O2, on cellular EPM. Under these conditions beta-radiation enhanced EPM response to aspirin only at concentration of 100 microM. The EPM response to BW755c is reduced by irradiation at all concentrations with the exception of equal-effective one (10 microM). Data obtained evidence for modification of eicosanoid metabolism by beta-radiation, probably, as a result of phospholipase A2 inhibition, as evident from elimination by radiation of stimulated action of hydrogen peroxide on EPM. The radiation action can also affect the cyclooxygenase lipoxygenase activity ratio, this activity being mediated by cellular membrane signaling systems.     

Status of the detoxication function of the liver after vagal denervation

In 14 and 25 days of bilateral subdiaphragmatic+ vagotomy perimeter of the endoplasmic reticulum (EPR) of hepatocytes, content of the enzyme (cytochrome R-450) built into the membrane in microsomal fractions, speed of hydroxylation of substrates and oxygen absorption decreased and the section area of EPR and duration of the hexonal sleep increased. The results demonstrate certain disturbances of the detoxication function of the denervated liver.     

Relationship between the structure of amphiphilic copolymers and their ability to disturb lipid bilayers

Nonionic amphiphiles and particularly block copolymers of ethylene oxide and propylene oxide (Pluronics) cause pronounced chemosensitization of tumor cells that exhibit multiple resistance to antineoplastic drugs. This effect is due to inhibition of P-glycoprotein (P-gp) responsible for drug efflux. It was suggested that the inhibition of P-gp might be due to changes in its lipid surrounding. Indeed, high dependence of P-gp activity on the membrane microviscosity was demonstrated [Regev et al. (1999) Eur. J. Biochem. 259, 18-24], suggesting that the ability of Pluronics to affect the P-gp activity is mediated by their effect on the membrane structure. We have found recently that adsorption of Pluronics on lipid bilayers induced considerable disturbance of the lipid packing [Krylova et al. (2003) Chemistry 9, 3930-3936]. In the present paper, we studied 19 amphiphilic copolymers, including newly synthesized hyperbranched polyglycerols, Pluronic and Brij surfactants, for their ability to accelerate flip-flop and permeation of antitumor drug doxorubicin (DOX) in liposomes. It was found that not only bulk hydrophobicity but also the chemical microstructure of the copolymer determines its membrane disturbing ability. Copolymers containing polypropylene oxide caused higher acceleration of flip-flop and DOX permeation than polysurfactants containing aliphatic chains. The effects of copolymers containing hyperbranched polyglycerol "corona" were more pronounced, as compared to the copolymers with linear poly(ethylene oxide) chains, indicating that a bulky hydrophilic block induces additional disturbances in the lipid bilayer. A good correlation between the copolymer flippase activity and a linear combination of copolymer bulk hydrophobicity and the van der Waals volume of its hydrophobic block was found. The relationship between the structure of a copolymer and its ability to disturb lipid membranes presented in this paper may be useful for the design of novel amphiphilic copolymers capable of affecting the activity of membrane transporters in living cells.     

<Emphasis Type="Italic">Lactobacillus fermentum</Emphasis> 90 TC-4 taxonomic status confirmation using whole genome sequencing and MALDI TOF mass spectrum

With the use of whole genome sequencing, the taxonomic status of Lactobacillus fermentum 90 TC-4 strain from Russian collections were studied. Complex analysis of phenotypical and genetic properties was conducted using phenotypic and molecular genetic methods. The main characteristics of the genome and biochemical activity profile of the strain were determined. A comparative analysis of the mass spectrum of ribosomal proteins of the strain, its biochemical properties, a fragment of 16S rRNA gene sequencing, and the entire genome revealed that the present strain belongs to the species L. fermentum, confirming its taxonomic status in accordance with modern taxonomy.     

Adsorption on synthetic membranes and immunogenic properties of aprotinin cross-linked by glutaraldehyde]

A procedure for the intermolecular crosslinking of aprotinin (natural polypeptide, an inhibitor of serine proteases) by glutaraldehyde was proposed. This autoconjugation of aprotinin increased its immunogenicity and the efficiency of sorption on nitrocellulose and polyvinylidene difluoride membranes. The immunization of animals by the autoconjugated aprotinin induced the production of antibodies that reacted with both conjugated and monomeric aprotinin. The properties of the crosslinked aprotinin are promising for the enhancement of sensitivity through its use in EIA and immunoblotting membrane methods that employ aprotinin and antiaprotinin antibodies.     

Control of apoptosis in influenza virus-infected cells by up-regulation of Akt and p53 signaling

PI3k-Akt and p53 pathways are known to play anti- and pro-apoptotic roles in cell death, respectively. Whether these pathways are recruited in influenza virus infection in highly productive monkey (CV-1) and canine (MDCK) kidney cells was studied here. Phosphorylation of Akt (Akt-pho) was found to occur only early after infection (5–9 h.p.i). Nuclear accumulation and phosphorylation of p53 (p53-pho), and expression of its natural target p21/waf showed low constitutive levels at this period, whereas all three parameters were markedly elevated at the late apoptotic stage (17–20 h.p.i.). Up-regulation of Akt-pho and p53-pho was not induced by UV-inactivated virus suggesting that it required virus replication. Also, mRNAs of p53 and its natural antagonist mdm2 were not increased throughout infection indicating that p53-pho was up-regulated by posttranslational mechanisms. However, p53 activation did not seem to play a leading role in influenza-induced cell death: (i) infection of CV1 and MDCK cells with recombinant NS1-deficient virus provoked accelerated apoptotic death characterized by the lack of p53 activation; (ii) mixed apoptosis-necrosis death developed in influenza-infected human bronchial H1299 cells carrying a tetracycline-regulated p53 gene did not depend on p53 gene activation by tetracycline. Virus-induced apoptosis and signaling of Akt and p53 developed in IFN-deficient VERO cells with similar kinetics as in IFN-competent CV1-infected cells indicating that these processes were endocrine IFN-independent. Apoptosis in influenza-infected CV-1 and MDCK cells was Akt-dependent and was accelerated by Ly294002, a specific inhibitor of PI3k-Akt signaling, and down-regulated by the viral protein NS1, an inducer of host Akt. The obtained data suggest that influenza virus (i) initiates anti-apoptotic PI3k-Akt signaling at early and middle phases of infection to protect cells from fast apoptotic death and (ii) provokes both p53-dependent and alternative p53-independent apoptotic and/or necrotic (in some host systems) cell death at the late stage of infection. These data have been partially presented at The 3rd Orthomyxovirus Research Conference (sponsored by ESWI and NIH). Abstr. p. 23 entitled: “Influenza virus-specific up-regulation of Akt and Mdm2 in infected cells” by Zhirnov O.P., and Klenk H.D., July 28–21, 2005. Queen’s College, Cambridge, United Kingdom; and at The Annual Meeting of Virology in Munich, March 15–18 (2006)—“Influenza virus-specific up-regulation of Akt, Mdm2, and p53 in infected cells” by O. P. Zhirnov and H. D. Klenk; Book of abstracts, p. 339     

Caspase-dependent N-terminal cleavage of influenza virus nucleocapsid protein in infected cells

The nucleocapsid protein (NP) (56 kDa) of human influenza A viruses is cleaved in infected cells into a 53-kDa form. Likewise, influenza B virus NP (64 kDa) is cleaved into a 55-kDa protein with a 62-kDa intermediate (O. P. Zhirnov and A. G. Bukrinskaya, Virology 109:174-179, 1981). We show now that an antibody specific for the N terminus of influenza A virus NP reacted with the uncleaved 56-kDa form but not with the truncated NP53 form, indicating the removal of a 3-kDa peptide from the N terminus. Amino acid sequencing revealed the cleavage sites ETD16*G for A/Aichi/68 NP and sites DID7*G and EAD61*V for B/Hong Kong/72 NP. With D at position -1, acidic amino acids at position -3, and aliphatic ones at positions -2 and +1, the NP cleavage sites show a recognition motif typical for caspases, key enzymes of apoptosis. These caspase cleavage sites demonstrated evolutionary stability and were retained in NPs of all human influenza A and B viruses. NP of avian influenza viruses, which is not cleaved in infected cells, contains G instead of D at position 16. Oligopeptide DEVD derivatives, specific caspase inhibitors, were shown to prevent the intracellular cleavage of NP. All three events, the NP cleavage, the increase of caspase activity, and the development of apoptosis, coincide in cells infected with human influenza A and B viruses. The data suggest that intracellular cleavage of NP is exerted by host caspases and is associated with the development of apoptosis at the late stages of infection.     

Sodium azide as indirect nitric oxide donor: researches on the rat aorta isolated segments

A comparative investigations of heme-containing enzymes inhibitors NaN3 and NaCN effects on the rat aorta isolated segments tone has shown that NaN3 in the range of very low concentrations from 10(-9) to 10(-6) M displays pharmacological activity characteristic of nitric oxide (NO) donors, which is inhibited by NaCN. The value of vasodilatation, caused by NaN3, was also decreased in the presence of soluble guanylate cyclase inhibitor ODQ (10(-5) M). It was found that H2O2 injection to physiological solution containing NaN3 and horseradish peroxidase or catalase lead to NO2- accumulation in it, which was blocked by NaCN. The nonenzymic NaN3 oxidization by hydrogen peroxide was not found in control experiments. NaN3 physiological activity dependent on NO-donating properties of this traditional inhibitor of heme-containing enzymes is discussed.     

Cleavage of influenza a virus hemagglutinin in human respiratory epithelium is cell associated and sensitive to exogenous antiproteases

Proteolytic cleavage of the hemagglutinin (HA) of human influenza viruses A/Aichi/2/68 (H3N2) and A/WSN/34 (H1N1) from HA0 to HA1/HA2 was studied in primary human adenoid epithelial cells (HAEC). HAEC contain a mixture of ciliated and nonciliated secretory cells and mimic the epithelium membrane of the human respiratory tract. Pulse-chase labeling with [(35)S]methionine and Western blot analysis with anti-HA antibodies of cellular and virion polypeptides showed that HAEC cleaved newly synthesized HA0 to HA1/HA2 ("cleavage from within") and significant amounts of cleaved HA accumulated within cells. It was also shown that HAEC was able to cleave HA0 of incoming virions ("cleavage from without"), whereas the HA0 of nonabsorbed virions free in extracellular fluid were not cleaved, supporting the conclusion that HA0 cleavage in HAEC is cell associated. Low-molecular-weight inhibitors of serine proteases, aprotinin and leupeptin, when added to influenza virus-infected HAEC suppressed HA0 cleavage and reduced the amount of cleaved HA1/HA2 both in cells and in progeny virions and thus diminished the infectivity of the virus. In contrast, the addition of fetal bovine serum, containing a number of high-molecular-weight antiproteases that compete for proteases in the extracellular environment, did not inhibit influenza virus growth in HAEC. These data suggest that in human respiratory epithelium the cleavage of influenza virus HA containing a single arginine in the proteolytic site (i) is a cell-associated process accomplished by serine-type protease(s) and (ii) is sensitive to low-molecular-weight exogenous inhibitors of serine proteases.