Biochemistry (Moscow) (v.78, #12)

Polyreactivity of natural antibodies: Exchange by HL-fragments by M. A. Sedykh; V. N. Buneva; G. A. Nevinsky (1305-1320).
The polyreactivity of binding (formation of antibody (AB) complexes not only with specific but also with foreign antigens) is a widespread phenomenon that in some cases can be caused by a conformational lability of the antigen-binding sites of antibodies (which increases upon treatment with various destabilizing agents) and leads to AB binding with very different antigens. Some ABs exist as dimers of the initial ABs and their idiotypes (or anti-idiotypes) capable of producing intramolecular cyclic complexes with features of polyreactants. Another mechanism of binding polyreactivity is an exchange in blood by halves of IgG4 molecules (HL-fragments) against various antigens. Also, for the first time catalytic polyfunctionality of human milk ABs has been detected, which is caused by an exchange by HL-fragments between molecules of λ- and κ-IgG (IgG1-IgG4) and also by λ- and κ-sIgA against different antigens with formation of very different chimeric antibodies. This review considers all possible pathways of formation of polyspecific immunoglobulins and their biological functions described in the literature, as well as mechanisms of binding polyreactivity and catalytic polyfunctionality of natural antibodies.
Keywords: antibodies; natural abzymes; polyreactivity of binding; catalytic polyfuctionality

This review analyzes agrobacterial virulence proteins and recipient cell proteins involved in horizontal transfer of a T-DNA-protein complex. Specifically, it considers the early stages of the interactions of partners (signal exchange, attachment, close contact); T-DNA release from bacterial cells; channel formation for the transfer of ssDNA between the partners; transfer of agrobacterial T-DNA through the membrane, cytoplasm, and nuclear membrane of the recipient cell and its incorporation into the recipient cell genome. It further discusses possible pathways of agrobacterial ssDNA transfer to the recipient cells. In particular, the possible role of T-pili and VirE2 protein during conjugative transfer of agrobacterial ssDNA between donor and recipient cells is discussed.
Keywords: horizontal transfer; Agrobacterium ; T-DNA; virulence proteins; conjugation; T-pili; VirE2

Lung cancer is a disease characterized by uncontrolled cell growth in tissues of the lung. Leptin is a pleiotropic hormone with antiapoptotic and proliferative roles involved in several systems. However, there is no known antiapoptotic mechanism of leptin in non-small cell lung cancer (NSCLC). So, we investigated the antiapoptotic mechanism of leptin in NSCLC. Proliferation, apoptosis, and the specific mechanism of leptin-transfected cells were analyzed in this study. Leptin, p-Perk, IRE1, cleaved ATF6, spliced XBP1, eIF2-α, TRAF2, CHOP, and caspase 12 proteins were detected by Western blot, and endoplasmic reticulum (ER) stress-related mRNA was detected by semi-quantitative reverse transcription PCR (RT-PCR). Leptin in A549 and transfected cells inhibited cisplatin-activated ER stress-associated mRNA transcription and activation of proteins. ER stress unfolded protein response (UPR) proteins, PERK and ATF6, were involved in leptin-triggered apoptosis. XBP1 and TRAF2 were increased significantly when treated with cisplatin in A549-siLPT and non-transfected cells. CHOP expression was blocked in A549 and transfected cells (LPT-PeP and LPT-EX cells). In conclusion, leptin can promote the proliferation of A549 cells through blocking ER stress-mediated apoptosis. This blocking is mediated by the p-Perk and ATF6 pathway through blocking activation of CHOP.
Keywords: apoptosis; ER stress; cell growth; leptin; TRAF2; XPB1

Effect of sex hormones on levels of mRNAs coding for proteins involved in lipid metabolism in macrophages by T. A. Shchelkunova; I. A. Morozov; P. M. Rubtsov; L. M. Samokhodskaya; I. V. Andrianova; E. G. Rudimov; I. A. Sobenin; A. N. Orekhov; A. N. Smirnov (1342-1353).
The effects of sex hormones estradiol (E2), testosterone (Te), and 5α-dihydrotestosterone (DT) on cholesterol accumulation induced by modified low density lipoproteins (LDL) in macrophages differentiated from human peripheral blood monocytes and on the levels of mRNAs coding for proteins involved in lipid metabolism have been studied. All three hormones at physiological concentrations (1 nM) are capable of reducing cholesterol accumulation in cells. The treatment of cells with modified and native (not inducing cholesterol accumulation) LDL results in similar alterations in the expression of several mRNAs aimed primarily at homeostatic regulation of lipid metabolism. These alterations depend on the sex of macrophage donors and in some cases are even reversed in cells obtained from male and female donors. The cells not treated with modified LDL have no significant gender differences in the expression of the examined mRNAs. Hormones, either independently or in combination with the modified LDL, influence the levels of some mRNAs, and each hormone shows an individual range of effects. Correlation analysis of changes in mRNA content in the cells showed that the hormones may interfere with coordination of gene expression. Hormone action leads to: (1) reduced coupling of the content of individual mRNAs with their initial levels in the control cells; (2) reduced coupling of different mRNA levels; (3) regrouping of mRNAs between the clusters; and (4) changes in the number of factors that determine the correlation links between mRNAs. The data show that sex hormones may have impact on the level of expression of certain genes and, in particular, on the coordination of gene expression in macrophages.
Keywords: sex hormones; PCR; gene expression; atherogenesis; macrophages; correlation analysis

Internal initiation of polyuridylic acid translation in bacterial cell-free system by E. A. Sogorin; S. Ch. Agalarov; A. S. Spirin (1354-1357).
The task of the present work was to answer the question: is the free 5′-end needed for effective translation of a model polyribonucleotide template — polyuridylic acid — in a bacterial (E. coli) cell-free system? For this purpose, the template activities of the original polyuridylic acid with its free 5′-end and the polyuridylic acid with blocked 5′-end were compared in the bacterial cell-free translation system. To block the 5′-end, the cytidylic oligodeoxyribonucleotide with fluorescein residue at its 5′-end and uridylic oligoribonucleotide sequence at its 3′-end, schematically described as FAM(dC)10(rU)50, was covalently attached (ligated) to the 5′-end of the template polyuridylic acid. It was shown that the efficiency of polyphenylalanine synthesis on the 5′-blocked template and on the polyuridylic acid with free 5′-end was virtually the same. It was concluded that bacterial ribosomes are capable of effectively initiating translation at the polyuridylic sequence independently of the 5′-end of template polyribonucleotide, i.e. via an internal initiation mechanism, in the absence of a Shine-Dalgarno sequence and AUG start codon.
Keywords: polyuridylic acid; translation initiation; polyphenylalanine synthesis; T4 RNA ligase; cell-free translation

This paper presents a new experimental approach for determining the individual optical characteristics of reduced heme a in bovine heart cytochrome c oxidase starting from a small selective shift of the heme a absorption spectrum induced by calcium ions. The difference spectrum induced by Ca2+ corresponds actually to a first derivative (differential) of the heme a 2+ absolute absorption spectrum. Such an absolute spectrum was obtained for the mixed-valence cyanide complex of cytochrome oxidase (a 2+ a 3 3+ -CN) and was subsequently used as a basis spectrum for further procession and modeling. The individual absorption spectrum of the reduced heme a in the Soret region was reconstructed as the integral of the difference spectrum induced by addition of Ca2+. The spectrum of heme a 2+ in the Soret region obtained in this way is characterized by a peak with a maximum at 447 nm and half-width of 17 nm and can be decomposed into two Gaussians with maxima at 442 and 451 nm and half-widths of ∼10 nm (589 cm−1) corresponding to the perpendicularly oriented electronic π→π* transitions B 0x and B 0y in the porphyrin ring. The reconstructed spectrum in the Soret band differs significantly from the “classical” absorption spectrum of heme a 2+ originally described by Vanneste (Vanneste, W. H. (1966) Biochemistry, 65, 838–848). The differences indicate that the overall γ-band of heme a 2+ in cytochrome oxidase contains in addition to the B 0x and B 0y transitions extra components that are not sensitive to calcium ions, or, alternatively, that the Vanneste’s spectrum of heme a 2+ contains significant contribution from heme a 3 2+ . The reconstructed absorption band of heme a 2+ in the α-band with maximum at 605 nm and half-width of 18 nm (850 cm−1) corresponds most likely to the individual Q 0y transition of heme a, whereas the Q 0x transition contributes only weakly to the spectrum.
Keywords: cytochrome c oxidase; Ca2+ ; heme a ; absorption spectrum; spectral shift

SkBQ — Prooxidant addressed to mitochondria by M. Y. Vyssokikh; B. V. Chernyak; L. V. Domnina; D. S. Esipov; O. Y. Ivanova; G. A. Korshunova; R. A. Symonyan; M. V. Skulachev; T. V. Zinevich; V. P. Skulachev (1366-1370).
Oxidative stress and mitochondrial dysfunction are the key links in the chain of development of pathologies associated with the violation of cellular energy metabolism. Development of mitochondria-addressed compounds highly specific for chemical processes is one of the most promising ways to develop approaches to the treatment of inherited and age-related diseases with mitochondrial etiology. Correlation of structure and chemical activity of the test compounds from a class of lipophilic cations revealed the key role of substituents in the aromatic ring of 1,4-benzoquinones in the manifestation of high antioxidant properties. In this work, it is shown that a synthesized benzoquinone derivative conjugated in position 6 with membrane-penetrating cation of decyltriphenylphosphonium and with substituents at position 2, 3, and 5 (SkBQ) has much lower antioxidant and significantly higher prooxidant activity in comparison with similar derivatives of plasto- and toluquinone SkQ1 and SkQT1 in experiments on isolated mitochondria. At the same time, SkBQ, like SkQ1 and SkQT1, can be reduced by the respiratory chain in the center i of complex III and decrease the mitochondrial membrane potential. In cell cultures of human fibroblasts, it was revealed that SkBQ does not protect cells from apoptosis induced by hydrogen peroxide. Under the same conditions, SkQ1 and SkQT1 exhibit a powerful protective effect. Thus, SkBQ can be seen as a mitochondria-addressed prooxidant. The possibility of using SkBQ as an anticancer drug for the treatment of cancers such as prostate cancer whose cells are sensitive to mitochondrial reactive oxygen species is discussed.
Keywords: mitochondria; reactive oxygen species; mitochondria-addressed anti- and prooxidants

Do YB2/0 cells produce alien sugars? by D. Quagliaroli (1371-1373).
Olovnikova et al. (“Impact on N-glycosylation profile of monoclonal anti-D antibodies as a way to control their immunoregulatory and cytotoxic properties” (2012) Biochemistry (Moscow), 77, 925–933) mentioned the presence of “alien sugars” on monoclonal antibodies (mAbs) produced by YB2/0 cell line. We summarize in this paper our previous findings on the glycosylation profile of two anti-D mAbs produced in this cell line (LFB-R297 and LFB-R593, so-called Roledumab). Our results show the absence of any immunogenic glycotopes, and furthermore neither immunogenicity nor other serious adverse reactions were observed during clinical trials.
Keywords: YB2/0; monoclonal antibodies; anti-D; glycosylation; immunogenicity

The prophylaxis of the hemolytic disease of the newborn — a mandatory procedure in obstetrics — requires significant amounts of plasma-derived polyclonal anti-D immunoglobulin. Despite numerous attempts, the proper technology for mass production of effective monoclonal anti-D is still not available. LFB Biotechnologies is currently performing clinical trials with recombinant anti-D antibody that has low fucose content and is expressed in the cells of rat myeloma YB2/0. It was shown that this drug is well tolerated, accelerates fast clearance of D+ red blood cells, and can inhibit anti-D immune response in Rhesus-negative volunteers.
Keywords: anti-D monoclonal antibodies; Roledumab; glycosylation; alloimmunization; clinical trials