Biochemistry (Moscow) (v.74, #12)

Nonviral vectors such as complexes of plasmid DNA with cationic lipids known as lipoplexes are considered as an attractive alternative to virus-based delivery systems. Unlike viruses, lipoplexes do not suffer from immunological and mutational hazards, though the efficiency of lipoplexes is often not sufficient for therapeutic purposes and require higher level of transfection than achieved until now. A number of critical steps responsible for transfection efficiency are discussed here. They include processes of lipoplexes formation, interaction with cell surface, their internalization into cell, and DNA release and delivery into the nucleus. All these processes should be thoroughly studied to be able to enhance the transfection efficacy.
Keywords: liposomes; cationic lipids; transfection; nonviral vectors; lipoplexes

Electrostatic binding of substituted metal phthalocyanines to enterobacterial cells: Its role in photodynamic inactivation by M. G. Strakhovskaya; Y. N. Antonenko; A. A. Pashkovskaya; E. A. Kotova; V. Kireev; V. G. Zhukhovitsky; N. A. Kuznetsova; O. A. Yuzhakova; V. M. Negrimovsky; A. B. Rubin (1305-1314).
The effect of ionic substituents in zinc and aluminum phthalocyanine molecules and of membrane surface charge on the interaction of dyes with artificial membranes and enterobacterial cells, as well as on photosensitization efficiency was studied. It has been shown that increasing the number of positively charged substituents enhances the extent of phthalocyanine binding to Escherichia coli cells. This, along with the high quantum yield of singlet oxygen generation, determines efficient photodynamic inactivation of Gram-negative bacteria by zinc and aluminum octacationic phthalocyanines. The effect of Ca2+ and Mg2+ cations and pH on photodynamic inactivation of enterobacteria in the presence of octacationic zinc phthalocyanine has been studied. It has been shown that effects resulting in lowering negative charge on outer membrane protect bacteria against photoinactivation, which confirms the crucial role in this process of the electrostatic interaction of the photosensitizer with the cell wall. Electrostatic nature of binding is consistent with mainly electrostatic character of dye interactions with artificial membranes of different composition. Lower sensitivity of Proteus mirabilis to photodynamic inactivation, compared to that of E. coli and Salmonella enteritidis, due to low affinity of the cationic dye to the cells of this species, was found.
Keywords: photodynamic inactivation; photosensitizer; phthalocyanines; enterobacteria; lipopolysaccharides; fluorescence quenching; fluorescence correlation spectroscopy

Vanillic acid as a novel specific inhibitor of snake venom 5′-nucleotidase: A pharmacological tool in evaluating the role of the enzyme in snake envenomation by B. L. Dhananjaya; A. Nataraju; C. D. Raghavendra Gowda; B. K. Sharath; C. J. M. D’souza (1315-1319).
Vanillic acid has been investigated for its inhibitory effect on Naja naja, Daboia russellii, and Trimeresurus malabaricus venom 5′-nucleotidase activity. Trimeresurus malabaricus venom 5′-nucleotidase activity was 1.3- and 8.0-fold higher than that of N. naja and D. russellii venoms, respectively. Substrate specificity studies showed that for all the venoms tested, 5′-AMP was the preferred substrate for 5′-nucleotidase. This indicates the central role of adenosine in snake envenomation. Vanillic acid selectively and specifically inhibited 5′-nucleotidase activity among several enzymes present in the three venoms tested. The inhibitor was competitive, as the inhibition was relieved by increased substrate concentration. It appears that the COOH group in vanillic acid is the determining factor for inhibition as vanillin, a structurally similar compound with respect to vanillic acid, had no inhibitory activity. This study for the first time exemplifies vanillic acid as a pharmacological tool in evaluating the role of 5′-nucleotidase in snake envenomation.
Keywords: 5′-nucleotidase; Naja naja ; Daboia russellii ; Trimeresurus malabaricus ; vanillic acid; purines

In the pathogenic Mycobacterium tuberculosis H37Rv, the causative agent of tuberculosis, the genetic and biochemical mechanisms for initiation of DNA replication are largely unknown. In the present study, we have characterized the physical interactions between M. tuberculosis DnaA and DnaB using both in vivo methods, such as bacterial two-hybrid assays, and in vitro techniques, such as surface plasmon resonance (SPR) and Pull-down/Western blotting. The full-length N-terminus (1–206 residues) of DnaB was found to interact with DnaA, while the shorter N-terminal domain of DnaB (1–125 residues), which lacked the linker region, did not. Further SPR and electrophoretic mobility shift assays indicated that the N-terminus (1–206 residues) of DnaB also had a critical role in regulating DnaA complex formation at the origin of replication (OriC). This regulatory effect was not obviously observed for DNA substrates containing only two DnaA-boxes. This is the first report showing a physical interaction between DnaA and replicative helicase DnaB from M. tuberculosis and the role in subsequent DnaA-OriC interactions. The findings reported here further the understanding of the regulatory mechanisms for initiation of DNA replication in this important human pathogen.
Keywords: Mycobacterium tuberculosis ; DnaB; replication origin; DnaA; OriC

C-terminal fragment of human laminin-binding protein contains a receptor domain for Venezuelan equine encephalitis and tick-borne encephalitis viruses by A. A. Malygin; E. I. Bondarenko; V. A. Ivanisenko; E. V. Protopopova; G. G. Karpova; V. B. Loktev (1328-1336).
Polyclonal and monoclonal antibodies (MABs) to human laminin-binding protein (LBP) can efficiently block the penetration of some alphaand flaviviruses into the cell. A panel of 13 types of MABs to human recombinant LBP was used for more detailed study of the mechanism of this process. Competitive analysis has shown that MABs to LBP can be divided into six different competition groups. MABs 4F6 and 8E4 classified under competition groups 3 and 4 can inhibit the replication of Venezuelan equine encephalitis virus (VEEV), which is indicative of their interaction with the receptor domain of LBP providing for binding with virions. According to enzyme immunoassay and immunoblotting data, polyclonal anti-idiotypic antibodies to MABs 4F6 and 8E4 modeling paratopes of the LBP receptor domain can specifically interact with VEEV E2 protein and tick-borne encephalitis virus (TBEV) E protein. Mapping of binding sites of MABs 4F6 and 8E4 with LBP by constructing short deletion fragments of the human LBP molecule has shown that MAB 8E4 interacts with the fragment of amino acid residues 187–210, and MAB 4F6 interacts with the fragment of residues 263–278 of LBP protein, which is represented by two TEDWS peptides separated by four amino acid residues. This suggested that the LBP receptor domain interacting with VEEV E2 and TBEV E viral proteins is located at the C-terminal fragment of the LBP molecule. A model of the spatial structure of the LBP receptor domain distally limited by four linear loops (two of which are represented by experimentally mapped regions of amino acid residues 187–210 and 263–278) as well as the central β-folded region turning into the α-helical site including residues 200–216 of the LBP molecule and providing for the interaction with the laminin-1 molecule has been proposed.
Keywords: laminin-binding protein; receptor domain; anti-idiotypic antibodies; peptide; TEDWS; Venezuelan equine encephalitis virus; tick-borne encephalitis virus

pH-dependent urea-induced unfolding of stem bromelain: Unusual stability against urea at neutral pH by B. Ahmad; G. M. Rathar; A. Varshney; R. H. Khan (1337-1343).
Equilibrium unfolding of stem bromelain (SB) with urea as a denaturant has been monitored as a function of pH using circular dichroism and fluorescence emission spectroscopy. Urea-induced denaturation studies at pH 4.5 showed that SB unfolds through a two-state mechanism and yields ΔG (free energy difference between the fully folded and unfolded forms) of ∼5.0 kcal/mol and C m (midpoint of the unfolding transition) of ∼6.5 M at 25°C. Very high concentration of urea (9.5 M) provides unusual stability to the protein with no more structural loss and transition to a completely unfolded state.
Keywords: stem bromelain; urea denaturation; protein stability

Bacterial synthesis, purification, and solubilization of membrane protein KCNE3, a regulator of voltage-gated potassium channels by S. A. Goncharuk; A. A. Shulga; Ya. S. Ermolyuk; P. K. Kuzmichev; V. A. Sobol; E. V. Bocharov; V. V. Chupin; A. S. Arseniev; M. P. Kirpichnikov (1344-1349).
An efficient method is described for production of membrane protein KCNE3 and its isotope labeled derivatives (15N-, 15N-/13C-) in amounts sufficient for structural-functional investigations. The purified protein preparation within different detergent micelles was characterized using dynamic light scattering, CD spectroscopy, and NMR spectroscopy. It is shown that within DPC/LDAO micelles the protein is in monomeric form and acquires mainly α-helical conformation. The existence of cross-peaks for all glycines of the 15N-HSQC NMR spectra as well as relatively small line widths (∼20 Hz) confirm the high quality of the preparation and the possibility of obtaining structural-dynamic information on KCNE3 by high resolution heteronuclear NMR spectroscopy.
Keywords: KCNE (MiRP); membrane protein; expression; purification; dynamic light scattering; NMR

Microbial origin of phenylcarboxylic acids in the human body by N. V. Beloborodov; A. S. Khodakova; I. T. Bairamov; A. Yu. Olenin (1350-1355).
In previous studies we demonstrated increased amounts of phenylcarboxylic acids (PCA) in serum of patients with sepsis. This observation prompted the present study of the ability of the human microbiome bacteria to produce PCA in vitro. PCA were detected in culture media by gas chromatography-mass spectrometry. Increased amounts of phenyllactic and p-hydroxyphenyllactic acids were produced by Klebsiella pneumonia, Escherichia coli, and Staphylococcus aureus. Certain strict anaerobes (bifidobacteria, lactobacteria, eubacteria) have also been found to actively produce these PCA, but these bacteria are not etiologically linked to sepsis. Thus our results demonstrate the ability of sepsis-related bacteria to produce PCA and provide experimental support for the theory that the accumulation of PCA in the blood of patients with sepsis results from microbial degradation of phenylalanine and tyrosine.
Keywords: phenylcarboxylic acids; phenyllactic acid; p-hydroxyphenyllactic acid; endogenous microflora; facultative and strict anaerobes; sepsis; gas chromatography-mass spectrometry

Recombinant TNF-binding protein from variola virus as a novel potential TNF antagonist by I. P. Gileva; T. S. Nepomnyashchikh; I. A. Ryazankin; S. N. Shchelkunov (1356-1362).
Gel-filtration chromatographic separation of the lysate of Sf21 insect cells infected with recombinant baculovirus BVi67 containing the gene for TNF-binding protein (CrmB) of variola virus (VARV) revealed that hTNF-cytotoxicity neutralization activity is associated with a fraction corresponding mainly to high molecular weight proteins (above 500 kDa) and less with fractions corresponding to proteins of 270 or 90 kDa. The recombinant VARV-CrmB protein has been purified by affinity chromatography. Difference in the experimentally determined and estimated (according to amino acid composition) VARV-CrmB molecular weight is due to glycosylation of the recombinant protein expressed in the insect cells. VARV-CrmB neutralizes in vitro the cytotoxic effect of hTNF and hLTα, and its TNF-neutralizing activity is two to three orders of magnitude higher compared to the analogous effects of type I and II soluble TNF receptors, comparable with the activity of mAb MAK195, and somewhat lower than the effect of the commercial drug Remicade.
Keywords: tumor necrosis factor; TNF-binding protein; variola virus; viroceptors; septic shock

Effect of solute hydrogen bonding capacity on osmotic stability of lysosomes by Shu-Jing Hao; Jun-Fang Hou; Guo-Jiang Zhang (1363-1367).
The effect of solute hydrogen bonding capacity on the osmotic stability of lysosomes was examined through measurement of free enzyme activity of lysosomes after their incubation in sucrose and poly(ethylene glycol) (PEG) (1500–6000 Da molecular mass) media. Free enzyme activity of the lysosomes was less in the PEG medium than that in the sucrose medium under the same hypotonic condition. The lysosomal enzyme latency loss decreased with increasing hydrogen bonding capacity of the solute. In addition, the lysosomes lost less latency at lower incubation temperature. The results indicate that solute hydrogen bonding capacity plays an important role in the osmotic protection of an incubation medium to lysosomes.
Keywords: hydrogen bonding capacity; lysosomal osmotic stability; lysosome

Heterogeneous set of cell wall teichoic acids in strains of Bacillus subtilis VKM B-760 and VKM B-764 by N. V. Potekhina; G. M. Streshinskaya; Yu. I. Kozlova; E. B. Kudryashova; S. N. Senchenkova; A. S. Shashkov; L. N. Anan’ina (1368-1374).
Cell walls of Bacillus subtilis VKM B-760 and VKM B-764 are characterized by heterogeneous composition of teichoic acids. Polymer I with structure -6)-β-D-Galp-(1→1)-sn-Gro-(3-P-, polymer II with structure -6)-α-D-Glcp-(1→1)-sn-Gro-(3-P-, and a small amount of unsubstituted 1,3-poly(glycerol phosphate) were detected in strain VKM B-760. Strain VKM B-764 contains an analogous set of teichoic acids, but a characteristic feature of polymer II is the presence of disubstituted glycerol residue with α-glucopyranose localization in the integral chain at C-1 hydroxyl and β-glucopyranose as a side branch at C-2 hydroxyl (polymer III): -6)-α-D-Glcp-(1→1)-[β-D-Glcp-(1→2)]-sn-Gro-(3-P-. The structures of polymer I in bacilli and polymer III in Gram-positive bacteria are described for the first time. Teichoic acids were studied by chemical methods and on the basis of combined analysis of one-dimensional 1H-, 13C-, and 31P-NMR spectra, homonuclear two-dimensional 1H/1H COSY, TOCSY, and ROESY, and heteronuclear two-dimensional 1H/13C gHSQC- and HMQC-TOCSY experiments. Simultaneous presence of several different structure teichoic acids in the bacillus cell walls as well as chemotaxonomical perspectives of the application of these polymers as species-specific markers for members of the Bacillus genus is discussed.
Keywords: Bacillus subtilis ; cell wall; teichoic acids; NMR spectroscopy

Melittin-regenerated purple membrane by Yue Zhang; Tao Su; Kun-sheng Hu (1375-1381).
We have investigated the character of melittin-regenerated purple membrane. Adding melittin to blue membrane causes the color transition and partial regeneration of the photocycle and the proton pump. The reconstitution of bacteriorhodopsin by melittin is proved to be charge-dependent. In studying the location of melittin binding on the blue membrane, we suggest that melittin anchors on the membrane through both hydrophobic and electrostatic interactions. The electro-static interaction is dominant. The binding sites for the electrostatic interaction should be on the surface of the membrane.
Keywords: bacteriorhodopsin; blue membrane; melittin; electrostatic interaction

The activities of inorganic pyrophosphatase (PPase) and adenosine triphosphatase (ATPase) were studied in the plasma membrane of Leishmania donovani promastigotes and amastigotes. It was shown that the specific activity of PPase was greater than that of ATPase in the promastigote plasma membrane. We characterized H+-PPase present in the plasma membrane of L. donovani and investigated its possible role in the survival of promastigote and amastigote. PPase activity was stimulated by K+ and sodium orthovanadate and inhibited by pyrophosphate analogs (imidodiphosphate and alendronate), KF, N,N′-dicyclohexylcarbodiimide (DCCD), thiol reagents (p-chloromercuribenzenesulfonate (PCMBS), N-ethylmaleimide (NEM), and phenylarsine oxide (PAO)), the ABC superfamily transport modulator verapamil, and also by the F1Fo-ATPase inhibitor quercetin. ATPase activity was stimulated by K+ and verapamil, inhibited by DCCD, PCMBS, NEM, sodium azide, sodium orthovanadate, and quercetin, and was unaffected by PAO. We conclude that there are significant differences within promastigote, amastigote, and mammalian host in cytosolic pH homeostasis to merit the inclusion of PPase transporter as a putative target for rational drug design.
Keywords: Leishmania donovani promastigote and amastigote; pyrophosphatase; ATPase; plasma membrane; imidodiphosphate; verapamil

Identification of complexes formed by ceruloplasmin with matrix metalloproteinases 2 and 12 by A. V. Sokolov; M. O. Pulina; K. V. Ageeva; O. S. Tcherkalina; E. T. Zakharova; V. B. Vasilyev (1388-1392).
Marked sensitivity to proteolytic degradation results in the loss of multiple antioxidant properties of ceruloplasmin (CP), the multicopper oxidase of mammalian plasma. In this study, gel filtration of virtually pure CP (purity 99.7%) yielded complexes of this protein. Subjecting the complexes to SDS-free PAGE revealed other proteins along with CP. These were identified as matrix metalloproteinases (MMP-2 and MMP-12) by means of tryptic fragment mass spectrometry. Electrophoretic bands corresponding to MMP-2 (72 and 67 kDa) and MMP-12 (22 kDa) displayed gelatinase activity. The identified proteinases contained heparin-binding motifs inherent in the complex-forming partners of CP, such as lactoferrin, myeloperoxidase, and serprocidines. Therefore, admixtures of MMPs can be efficiently eliminated from CP preparations by chromatography on heparin-Sepharose as proposed previously.
Keywords: ceruloplasmin; matrix metalloproteinases; proteolysis

We identified a novel human-specific family of transposable elements that consists of fused copies of the CpG-island containing the first exon of gene MAST2 and retrotransposon SVA. We propose a mechanism for the formation of this family termed CpG-SVA, comprising 5′-transduction by an SVA insert. After the divergence of human and chimpanzee ancestor lineages, retrotransposon SVA has inserted into the first intron of gene MAST2 in the sense orientation. Due to splicing of an aberrant RNA driven by MAST2 promoter, but terminally processed using SVA polyadenylation signal, the first exon of MAST2 has fused to a spliced 3′-terminal fragment of SVA retrotransposon. The above ancestor CpG-SVA element due to retrotranspositions of its own copies has formed a novel family represented in the human genome by 76 members. Recruitment of a MAST2 CpG island was most likely beneficial to the hybrid retrotransposons because it could significantly increase retrotransposition frequency. Also, we show that human L1 reverse transcriptase adds an extra cytosine residue to the 3′ terminus of the nascent first strand of cDNA.
Keywords: molecular evolution; human DNA; retroelements; genomic transposable elements; regulation of transcription

Much evidence has recently been reported suggesting that reactive oxygen species (ROS) produced in mitochondria play a crucial role in the programmed senescence of organisms. In particular, it has been shown that antioxidants addressed to mitochondria slow down the appearance of symptoms of senescence and development of senile diseases and increase the median lifespan of various organisms from fungi to mammals. At the biochemical level, the mechanism of action of such rechargeable antioxidants as plastoquinonyldecyltriphenyl phosphonium (SkQ1) includes, in particular, prevention of oxidation of mitochondrial cardiolipin by ROS. The hormone melatonin also exhibits a number of such effects, and decrease in its level with age could explain the weakening of antioxidant protection upon aging. According to Moosmann et al., there exists a natural mechanism of antioxidant protection that, like SkQ1, is localized in the internal mitochondrial membrane and is rechargeable. It involves methionine residues in the surface regions of proteins encoded by mitochondrial DNA. It appears that in organisms with high respiratory metabolism the genetic code in the mitochondrial system of protein biosynthesis has changed. In these organisms (including some yeasts, insects, crustaceans, and vertebrates), the AUA codon codes for methionine rather than isoleucine, as in the case of synthesis of proteins encoded either in the nucleus or in mitochondria of organisms with lower rates of metabolism (other yeast species, sponges, and echinoderms). Methionine quenches ROS, being converted to methionine sulfoxide, which is re-reduced to the initial methionine by NADPH.
Keywords: programmed senescence; reactive oxygen species; mitochondria; antioxidants; methionine; mitochondrial genetic code

A hypothetical mechanism for rapid and nonrandom emergence of evolutionary adaptations is proposed. It is supposed that some transcription factors and transcription regulators that are able to cross membranes can leave the cells of their origin and move within the organism using a specialized transport system when individual development occurs under conditions extreme for the given species. This system, in particular, connects soma with germline. The supply of germline cells with unusual transcription regulators changes the balance of their nuclear regulatory RNAs, thus initiating RNA-dependent epigenetic modifications of the germline genome and therefore changes in phenotypes of the progeny. It is highly probable that some of these phenotypes are adaptive and lay the basis for the origin of the next biological species. The proposed mechanism can serve as a basis for a new theory of the origin of species.
Keywords: transcription factors; intercellular communications; speciation