Biochemistry (Moscow) (v.74, #10)

Critical evaluation of toxic versus beneficial effects of methylglyoxal by D. Talukdar; B. S. Chaudhuri; M. Ray; S. Ray (1059-1069).
In various organisms, an array of enzymes is involved in the synthesis and breakdown of methylglyoxal. Through these enzymes, it is intimately linked to several other physiologically important metabolites, suggesting that methylglyoxal has some important role to play in the host organism. Several in vitro and in vivo studies showed that methylglyoxal acts specifically against different types of malignant cells. These studies culminated in a recent investigation to evaluate a methylglyoxal-based formulation in treating a small group of cancer patients, and the results were promising. Methylglyoxal acts against a number of pathogenic microorganisms. However, recent literature abounds with the toxic effects of methylglyoxal, which are supposed to be mediated through methylglyoxal-derived advanced glycation end products (AGE). Many diseases such as diabetes, cataract formation, hypertension, and uremia are proposed to be intimately linked with methylglyoxal-derived AGE. However methylglyoxal-derived AGE formation and subsequent pathogenesis might be a very minor event because AGE are nonspecific reaction products that are derived through the reactions of carbonyl groups of reducing sugars with amino groups present in the side chains of lysine and arginine and in terminal amino groups of proteins. Moreover, the results of some in vitro experiments with methylglyoxal under non-physiological conditions were extrapolated to the in vivo situation. Some experiments even showed contradictory results and were differently interpreted. For this reason conclusions about the potential beneficial effects of methylglyoxal have often been neglected, thus hindering the advancement of medical science and causing some confusion in fundamental understanding. Overall, the potential beneficial effects of methylglyoxal far outweigh its possible toxic role in vivo, and it should be utilized for the benefit of suffering humanity.
Keywords: methylglyoxal; advanced glycation end products; glucose; cancer; antiviral

Dendrimers in gene transfection by D. G. Shcharbin; B. Klajnert; M. Bryszewska (1070-1079).
Dendrimers are a new class of nanocomposite materials. They are branching polymers whose structure is formed by monomeric subunit branches diverging to all sides from a central nucleus. The type of nucleus, attached monomers, and functional groups can be chosen during synthesis, which produces dendrimers of definite size, shape, density, polarity, branch mobility, and solubility. This review deals with problems of dendrimer molecular structures and capability of in vitro, in vivo, ex vivo, and in situ transfection of genetic material. Advantages and shortcomings of different types of dendrimers in this respect are discussed.
Keywords: dendrimer; complex; transfection; gene; DNA; RNA

Reduction of photosystem I reaction center by recombinant DrgA protein in isolated thylakoid membranes of the cyanobacterium Synechocystis sp. PCC 6803 by I. V. Elanskaya; V. A. Toporova; V. G. Grivennikova; E. M. Muronets; E. P. Lukashev; K. N. Timofeev (1080-1087).
To study the function of soluble NAD(P)H:quinone oxidoreductase of the cyanobacterium Synechocystis sp. PCC 6803 encoded by drgA gene, recombinant DrgA protein carrying 12 histidine residues on the C-terminal end was expressed in Escherichia coli and purified. Recombinant DrgA is a flavoprotein that exhibits quinone reductase and nitroreductase activities with NAD(P)H as the electron donor. Using EPR spectroscopy, it was demonstrated that addition of recombinant DrgA protein and NADPH to DCMU-treated isolated thylakoid membranes of the cyanobacterium increased the dark rereduction rate of the photosystem I reaction center (P700+). Thus, DrgA can participate in electron transfer from NADPH to the electron transport chain of the Synechocystis sp. PCC 6803 thylakoid membrane.
Keywords: cyanobacteria; drgA gene; NAD(P)H:quinone oxidoreductase; cyclic electron transport around photosystem I; EPR spectroscopy

The effects of pH on Clitoria ternatea agglutinin (CTA) were studied by spectroscopy, size-exclusion chromatography, and by measuring carbohydrate specificity. At pH 2.6, CTA lacks well-defined tertiary structure, as seen by fluorescence and near-UV CD spectra. Far-UV CD spectra show retention of 50% native-like secondary structure. The mean residue ellipticity at 217 nm plotted against pH showed a transition around pH 4.0 with loss of secondary structure leading to the formation of an acid-unfolded state. This state is relatively less denatured than the state induced by 6 M guanidine hydrochloride. With a further decrease in pH, this unfolded state regains ∼75% secondary structure at pH 1.2, leading to the formation of the A-state with native-like near-UV CD spectral features. Enhanced 8-anilino-1-naphthalene-sulfonate binding was observed in A-state, indicating a “molten-globule” like conformation with exposed hydrophobic residues. Acrylamide quenching data exhibit reduced accessibility of quencher to tryptophan, suggesting a compact conformation at low pH. Size-exclusion chromatography shows the presence of a compact intermediate with hydrodynamic size corresponding to a monomer. Thermal denaturation of the native state was cooperative single-step transition and of the A-state was non-cooperative two-step transition. A-State regains 72% of the carbohydrate-binding activity.
Keywords: acid-induced unfolding; Clitoria ternatea agglutinin; carbohydrate binding; molten-globule state; thermal stability

Effects of entomocidal Cry-type proteins, δ-endotoxins Cry3A and Cry11A produced by Bacillus thuringiensis, on ion permeability of the apical membranes of intestinal epithelium from Tenebrio molitor larvae midgut were studied. Using potential-sensitive dyes safranine O and oxonol VI and δpH indicator acridine orange, it was shown that placing brush border membrane vesicles (BBMV) (loaded with Mg2+ during their preparation) into a salt-free buffer medium resulted in spontaneous generation of transmembrane electric potential on the vesicular membrane (negative inside the vesicles) accompanied by acidification of the aqueous phase inside the vesicles. The generation of transmembrane ion gradients on the vesicular membrane was a result of an electrogenic efflux of Mg2+ from the vesicles as shown by abolishing of the membrane potential by such agents as MgSO4 or CaCl2 in centimolar concentrations, a highly lipophilic cation tetraphenylphosphonium, and some blockers of cell membrane Ca2+-channels in submillimolar concentrations. A passive generation of membrane potential on the vesicular membrane (but positive inside the vesicles) was also observed upon addition of centimolar concentrations of K2SO4. Addition of δ-endotoxins Cry3A and Cry11A to the vesicle suspension in a salt-free buffer medium or in the same medium supplemented with centimolar concentrations of K2SO4 exerted a pronounced hyperpolarization of the vesicular membrane. This hyperpolarization was sensitive to the same agents, which abolished the membrane potential generation in the absence of δ-endotoxin. It is concluded that Cry proteins induced in BBMV from T. molitor opening pores or ion channels, which were considerably more permeable for alkaline- and alkaline-earth metal cations than for the accompanying anions.
Keywords: Bacillus thuringiensis ; δ-endotoxin Cry3A; Tenebrio molitor ; BBMV; membrane potential; ion channels; potential-sensitive dyes; membranotropic effect of δ-endotoxin Cry3A

Inhibitory effect of angiostatins on activity of the plasminogen/plasminogen activator system by R. B. Aisina; L. I. Mukhametova; D. A. Gulin; M. Y. Levashov; N. V. Prisyazhnaya; K. B. Gershkovich; S. D. Varfolomeyev (1104-1113).
Angiostatins, kringle-containing fragments of plasminogen, are potent inhibitors of angiogenesis. Effects of three angiostatin forms, K1–3, K1–4, and K1-4.5 (0–2 µM), on the rate of native Glu-plasminogen activation by its physiological activators in the absence or presence of soluble fibrin were investigated in vitro. Angiostatins did not affect the intrinsic amidolytic activities of plasmin and plasminogen activators of tissue type (tPA) and urokinase type (single-chain scuPA and two-chain tcuPA), but inhibited conversion of plasminogen to plasmin in a dose-dependent manner. All three angiostatins suppressed Glu-plasminogen activation by tcuPA independently of the presence of fibrin, and the inhibitory effect increased in the order: K1-3 < K1-4 < K1-4.5. The inhibitory effects of angiostatins on the scuPA activator activity were lower and further decreased in the presence of fibrin. Angiostatin K1-3 (up to 2 µM) had no effect, while 2 µM angiostatins K1-4 and K1-4.5 inhibited the fibrin-stimulated Glu-plasminogen activation by tPA by 50 and 100%, respectively. The difference in effects of the three angiostatins on the Glu-plasminogen activation by scuPA, tcuPA, and tPA in the absence or presence of fibrin is due to the differences in angiostatin structures, mechanisms of action, and fibrin-specificity of plasminogen activators, as well as due to the influence of fibrin on the Glu-plasminogen conformation. Angiostatins in vivo, which mimic plasminogen-binding activity, can inhibit plasminogen activation stimulated by various proteins (including fibrin) of extracellular matrix, thereby blocking cell migration and angiogenesis. The data of this work indicate that the inhibition of Glu-plasminogen activation under the action of physiological plasminogen activators by angiostatins can be implicated in the complex mechanism of their antiangiogenic and antitumor action.
Keywords: plasminogen; plasminogen activator; plasmin; angiostatin; inhibition of plasminogen activation

Molecular mechanisms of transformation of SkQ mitotropic quinones and the search for new approaches to creation of selective free radical traps by S. A. Eremeyev; V. I. Kargin; K. A. Motovilov; V. N. Tashlitsky; V. Yu. Markov; G. A. Korshunova; N. V. Sumbatyan; M. Yu. Vyssokikh; L. S. Yaguzhinsky (1114-1124).
Features of the mechanism of action of positively charged benzoquinone derivatives (SkQ), which are the analogs of coenzyme Q (I), plastoquinone (II), and tocopherol (III), are discussed. It is usually considered that the main target of these compounds is mitochondria, where they accumulate due to the positive charge of the molecule. In the present work, it is shown with model systems that the reduced forms of compounds (I–III) under certain conditions can transform into electrically neutral cyclic zwitterions, which theoretically can escape from the matrix of energized mitochondria against the concentration gradient. A weak uncoupling effect of molecules I–III has been found on mitochondria. Its existence is in agreement with the abovementioned transformation of positively charged hydroquinones of type Ia–IIIa into electrically neutral molecules. The data obtained with model systems suggest that the target of SkQ hydroquinones as free radical traps may be not only mitochondria but also biochemical systems of the cytoplasm. Due to the presence of a large number of reactive oxygen species (ROS)-dependent signal systems in a cell, the functioning of cytoplasmic systems might be disturbed under the action of antioxidants. The problem of selective effect of antioxidants is discussed in detail in the present work, and a functional diagram of selective decrease of the “background level” of ROS based on differences in the intensity of background and “signal” ROS fluxes is considered.
Keywords: free radical traps; mitochondria; ROS; SkQ; MitoQ; uncoupling

Lignin peroxidase from the liquid culture filtrate of Gloeophyllum sepiarium MTCC-1170 has been purified to homogeneity. The molecular weight of the purified enzyme was 42 kDa as determined by SDS-PAGE. The K m values were 54 and 76 µM for veratryl alcohol and H2O2, respectively. The pH and temperature optima were 2.5 and 25°C, respectively. Depolymerization of coal by the fungal strain has been demonstrated using humic acid as a model of coal. Depolymerization of humic acid by the purified lignin peroxidase has been shown by the decrease in absorbance at 450 nm and increase in absorbance at 360 nm in presence of H2O2. Depolymerization of humic acid by the purified enzyme has also been demonstrated by the decrease in the viscosity with time of the reaction solution containing humic acid, H2O2, and the purified lignin peroxidase. The influence of NaCl and NaN3 and inhibitory effects of various metal chelating agents on the lignin peroxidase activity were studied.
Keywords: lignin peroxidase; Gloeophyllum sepiarium ; veratryl alcohol; humic acid

Structure-function analysis of mutant RNA-dependent RNA polymerase complexes with VPg by Chaojiang Gu; Tao Zeng; Yong Li; Zhenghui Xu; Zhongxi Mo; Congyi Zheng (1132-1141).
The replication of the foot-and-mouth disease virus (FMDV) genome is critically dependent upon the activity of a virally encoded RNA-dependent RNA polymerase (RdRp). In this study, four mutant RdRps of FMDV were isolated from viral quasi-species treated with ribavirin, of which two were single mutants (L123F and T381A) and two were double mutants (T291I/T381I and L123F/F244L). The mutant proteins were expressed in Escherichia coli and purified by His-bind resin chromatography. In combination with real-time RT-PCR, an in vitro RNA replication system that uses genome RNA/VPg as template-primers was used to determine polymerase activity. Mutant L123F exhibited a 0.6-fold decrease (p < 0.001) in polymerase activity relative to wild-type RdRp, whereas the activity of L123F/F244L and T381A was undetectable. Surprisingly, the activity of T291I/T381I yielded a 0.7-fold increase (p < 0.001) as compared to wild-type. In order to study the structure-function relationship of RdRp, all structures of the RdRp-RNA template-primer complex were obtained through homology modeling and molecular docking. The VPg1 orientation in the RdRp-VPg1 complexes was determined and analyzed with mathematical methods. Our results reveal that the orientation of VPg after binding to the polymerase determines the FMDV RdRp catalytic activity, which provides a basis for the rational design of novel antiviral agents.
Keywords: foot-and-mouth disease virus; RNA-dependent RNA polymerase activity; homogenous modeling; molecular docking; VPg orientation

Bacterial production and refolding from inclusion bodies of a “Weak” toxin, a disulfide rich protein by E. N. Lyukmanova; M. A. Shulepko; R. V. Tikhonov; Z. O. Shenkarev; A. S. Paramonov; A. N. Wulfson; I. E. Kasheverov; T. L. Ustich; Yu. N. Utkin; A. S. Arseniev; V. I. Tsetlin; D. A. Dolgikh; M. P. Kirpichnikov (1142-1149).
The gene for the “weak” toxin of Naja kaouthia venom was expressed in Escherichia coli. “Weak” toxin is a specific inhibitor of nicotine acetylcholine receptor, but mechanisms of interaction of similar neurotoxins with receptors are still unknown. Systems previously elaborated for neurotoxin II from venom of the cobra Naja oxiana were tested for bacterial production of “weak” toxin from N. kaouthia venom. Constructs were designed for cytoplasmic production of N. kaouthia “weak” toxin in the form of a fused polypeptide chain with thioredoxin and for secretion with the leader peptide STII. However, it became possible to obtain “weak” toxin in milligram amounts only within cytoplasmic inclusion bodies. Different approaches for refolding of the toxin were tested, and conditions for optimization of the yield of the target protein during refolding were investigated. The resulting protein was characterized by mass spectrometry and CD and NMR spectroscopy. Experiments on competitive inhibition of 125I-labeled α-bungarotoxin binding to the Torpedo californica electric organ membranes containing the muscle-type nicotine acetylcholine receptor (α12β1γδ) showed the presence of biological activity of the recombinant “weak” toxin close to the activity of the natural toxin (IC50 = 4.3 ± 0.3 and 3.0 ± 0.5 µM, respectively). The interaction of the recombinant toxin with α7 type human neuronal acetylcholine receptor transfected in the GH4C1 cell line also showed the presence of activity close to that of the natural toxin (IC50 31 ± 5.0 and 14.8 ± 1.3 µM, respectively). The developed bacterial system for production of N. kaouthia venom “weak” toxin was used to obtain 15N-labeled analog of the neurotoxin.
Keywords: neurotoxins; nicotine acetylcholine receptor; bacterial expression; refolding

High-resolution atomic force microscopy of DNA by D. V. Klinov; T. V. Neretina; V. V. Prokhorov; T. V. Dobrynina; K. G. Aldarov; V. V. Demin (1150-1154).
A method using high resolution atomic force microscopy for imaging DNA has been elaborated. Using super-sharp probes and modified graphite as support for molecule adsorption, DNA molecule images were obtained whose resolution made possible the observation of their fine structure with repeated helical motifs. The method can be used to visualize individual spread molecules of single-stranded DNA.
Keywords: AFM; high resolution; DNA; scanning probes; HOPG

Pma1 H+-ATPase is the primary proton pump in the plasma membrane of the yeast Saccharomyces cerevisiae. It generates an electrochemical proton gradient, thus providing energy for secondary solute transport systems. The enzyme contains nine cysteines, three (Cys148, Cys312, and Cys867) in transmembrane segments and the rest (Cys221, Cys376, Cys409, Cys472, Cys532, and Cys569) in the cytosolic parts of the molecule. Although individually they are not essential for the functioning of the ATPase, substitution of all of them leads to the loss of enzyme activity and impairment of biogenesis. By means of site-directed mutagenesis combined with other molecular-biological and biochemical methods, this work defines different combinations of minimal cysteine content that are consistent with ATPase function.
Keywords: ATPase; plasma membrane; secretory vesicles; yeast; cysteine; site-directed mutagenesis

Oligopeptidase B from Serratia proteamaculans. I. Determination of primary structure, isolation, and purification of wild-type and recombinant enzyme variants by R. F. Khairullin; A. G. Mikhailova; T. Yu. Sebyakina; N. L. Lubenets; R. H. Ziganshin; I. V. Demidyuk; T. Yu. Gromova; S. V. Kostrov; L. D. Rumsh (1164-1172).
A novel trypsin-like protease (PSP) from the psychrotolerant gram-negative microorganism Serratia proteamaculans was purified by ion-exchange chromatography on Q-Sepharose and affinity chromatography on immobilized basic pancreatic trypsin inhibitor (BPTI-Sepharose). PSP formed a tight complex with GroEL chaperonin. A method for dissociating the GroEL-PSP complex was developed. Electrophoretically homogeneous PSP had molecular mass of 78 kDa; the N-terminal amino acid sequence 1–10 was determined, and mass-spectral analysis of PSP tryptic peptides was carried out. The enzyme was found to be the previously unknown oligopeptidase B (OpdB). The S. proteamaculans 94 OpdB gene was sequenced and the producer strain Escherichia coli BL-21(DE3) pOpdB No. 22 was constructed. The yield of expressed His6-PSP was 1.5 mg/g biomass.
Keywords: oligopeptidase B; trypsin; Serratia proteamaculans ; GroEL; fusion expression