Biochemistry (Moscow) (v.77, #12)
Methods for in vivo molecular imaging by A. A. Kuchmiy; G. A. Efimov; S. A. Nedospasov (1339-1353).
Visualization of single molecules and specific subsets of cells is widely used for studies of biological processes and particularly in immunological research. Recent technological advances have provided a qualitative change in biological visualization from studying of “snapshot” pictures to real-time continuous observation of cellular dynamics in vivo. Contemporary methods of in vivo imaging make it possible to localize specific cells within organs and tissues, to study their differentiation, migration, and cell-to-cell interactions, and to follow some intracellular events. Fluorescence intravital microscopy plays an especially important role in high resolution molecular imaging. The methods of intravital microscopy are quickly advancing thanks to improvements in molecular sensors, labeling strategies, and detection approaches. Novel techniques allow simultaneous detection of various probes with better resolution and depth of imaging. In this review, we describe current methods for in vivo imaging, with special accent on fluorescence approaches, and discuss their applications for medical and biological studies.
Keywords: in vivo visualization; fluorescence; microscopy; label; reporter mice
Cytokinin oxidase is key enzyme of cytokinin degradation by A. M. Avalbaev; K. A. Somov; R. A. Yuldashev; F. M. Shakirova (1354-1361).
Cytokinin oxidase (EC 126.96.36.199) is an enzyme that catalyzes the irreversible degradation of cytokinin phytohormones that are extremely necessary for growth, development, and differentiation of plants. Cytokinin oxidase plays an important role in the regulation of quantitative level of cytokinins and their distribution in plant tissues. This review generalizes the available information on the structure, properties, and functional role of this enzyme in plant ontogeny under conditions of normal growth and under the influence of unfavorable environmental factors.
Keywords: cytokinin oxidase; cytokinins; cytokinin degradation
Application of recombinant antibody fragments for troponin I measurements by E. P. Altshuler; A. V. Vylegzhanina; I. A. Katrukha; A. V. Bereznikova; D. V. Serebryanaya (1362-1367).
The cardiac isoform of troponin I is a reliable biomarker of damaged cardiomyocytes that accompanies such severe cardiovascular diseases as myocardial infarction. Monoclonal antibody 19C7 recognizes troponin I in the blood-stream with high affinity and specificity. Recombinant antibodies can be used to improve detection systems based on monoclonal antibodies produced with hybridoma technology. In the present study, we compare the properties of monoclonal anti-body 19C7 and its recombinant fragments. It is shown that the recombinant antibody fragments demonstrate similar affinity values as monoclonal antibodies and can be applied for troponin I detection.
Keywords: monoclonal antibodies; recombinant antibodies; troponin I; affinity; fluorescence immunoassay; biotinylation
Iron-dependent superoxide dismutase from novel thermoacidophilic crenarchaeon Acidilobus saccharovorans: from gene to active enzyme by E. S. Slutskaya; E. Yu. Bezsudnova; A. V. Mardanov; I. V. Safenkova; S. Yu. Kleimenov; N. A. Chebotareva; V. M. Gumerov; N. V. Ravin; K. G. Skryabin; V. O. Popov (1368-1376).
A gene encoding superoxide dismutase was revealed in the genome of the thermoacidophilic crenarchaeon Acidilobus saccharovorans. A recombinant expression vector was constructed and transformed into E. coli cells. The novel recombinant superoxide dismutase was purified and characterized. The enzyme was shown to be an iron-dependent super-oxide dismutase able to bind various bivalent metals in the active site. According to differential scanning calorimetric data, the denaturation temperature of the enzyme is 107.3°C. The maximal activity of the Fe(II) reconstituted enzyme defined by xanthine oxidase assay is 1700 U/mg protein. Study of the thermal stability of the superoxide dismutase samples with various metal contents by tryptophan fluorescence indicated that the thermal stability and activity of the enzyme directly depend on the nature of the reconstituted metal and the degree of saturation of binding sites.
Keywords: Acidilobus saccharovorans ; metalloenzyme; Fe-dependent superoxide dismutase; thermal stability
Interaction of the synthetic peptide octarphin with rat adrenal cortex membranes by Y. N. Nekrasova; Y. A. Zolotarev; E. V. Navolotskaya (1377-1381).
The synthetic peptide octarphin (TPLVTLFK, fragment 12–19 of β-endorphin), a selective agonist of the nonopioid β-endorphin receptor, was labeled with tritium yielding specific activity of 28 Ci/mmol. The binding of [3H]octarphin to rat adrenal cortex membranes was studied under normal conditions as well as after cold and heat shocks. It was found that under normal conditions [3H]octarphin specifically binds to the membranes with high affinity: K d1 = 36.3 ± 2.5 nM, Bmax1 = 41.0 ± 3.8 pmol/mg protein. The specific binding of [3H]octarphin to the membranes was inhibited by unlabeled β-endorphin (K i = 33.9 ± 3.6 nM) and the agonist of the non-opioid receptor decapeptide immunorphin (K i = 36.8 ± 3.3 nM). Unlabeled naloxone, [Leu5]- and [Met5]enkephalins, α- and γ-endorphins, and corticotropin were inactive (K i > 1 μM). Both cold and heat shocks decreased the binding affinity: K d2 = 55.6 ± 4.2 nM and K d3 = 122.7 ± 5.6 nM, respectively. In both cases, the maximal binding capacity of the receptor did not change. Thus, even a short-term thermal shock significantly affects the sensitivity of the non-opioid β-endorphin receptor of adrenal cortex membranes.
Keywords: β-endorphin; naloxone; peptides; receptors; adrenal cortex
Effect of plastoquinone derivative 10-(6′-Plastoquinonyl) decyltriphenylphosphonium (SkQ1) on estrous cycle and 17β-estradiol level in rats by V. A. Chistyakov; S. V. Dem’yanenko; A. A. Alexandrova; L. V. Gutnikova; V. N. Prokof’ev; O. N. Kosheleva (1382-1386).
Administration of the plastoquinone derivative 10-(6′-plastoquinonyl)decyltriphenylphosphonium (SkQ1) to female Wistar rats with regular estrous cycle once a day for two weeks at doses of 25 nmol/kg (but not 250 nmol/kg) leads to increase in proestrus duration by reducing the phase of diestrus and metestrus. Neither dose caused significant changes in serum 17β-estradiol level for any stage of the cycle. However, relative elongation of the proestrus stage leads to an increase in average per cycle estradiol levels by 20%.
Keywords: SkQ1; penetrating cations; estradiol; estrous cycle
Receptor-binding domain of ephrin-A1: Production in bacterial expression system and activity by O. V. Nekrasova; G. V. Sharonov; R. V. Tikhonov; P. M. Kolosov; M. V. Astapova; S. A. Yakimov; A. I. Tagvey; A. A. Korchagina; O. V. Bocharova; A. N. Wulfson; A. V. Feofanov; M. P. Kirpichnikov (1387-1394).
Eph receptor tyrosine kinases and their ligands, the ephrins, perform an important regulatory function in tissue organization, as well as participate in malignant transformation of cells. Ephrin-A1, a ligand of A class Eph receptors, is a modulator of tumor growth and progression, and the mechanism of its action needs detailed investigation. Here we report on the development of a system for bacterial expression of an ephrin-A1 receptor-binding domain (eA1), a procedure for its purification, and its renaturation with final yield of 50 mg/liter of culture. Functional activity of eA1 was confirmed by immunoblotting, laser scanning confocal microscopy, and flow cytometry. It is shown that monomeric non-glycosylated receptor-binding domain of ephrin-A1 is able to activate cellular EphA2 receptors, stimulating their phosphorylation. Ligand eA1 can be used to study the features of ephrin-A1 interactions with different A class Eph receptors. The created expression cassette is suitable for the development of ligands with increased activity and selectivity and experimental systems for the delivery of cytotoxins into tumor cells that overexpress EphA2 or other class A Eph receptors.
Keywords: ephrin-A1; receptor-binding domain; renaturation; HPLC; EphA2 receptor
Laccases: Complex architectures for one-electron oxidations by A. C. Mot; R. Silaghi-Dumitrescu (1395-1407).
Laccase (p-diphenol:dioxygen oxidoreductase), one of the earliest discovered enzymes, contains four copper ions in two active sites and catalyzes a one-electron oxidation of substrates such as phenols and their derivatives, or aromatic amines, coupled to a four-electron reduction of dioxygen to water. The catalytic mechanism has been studied for decades but is still not completely elucidated, especially in terms of the reduction of dioxygen to water. The key structural features of this enzyme are under investigation in several groups using techniques such as X-ray diffraction, electron paramagnetic resonance (EPR) spectroscopy, and site-directed mutagenesis. The high interest in laccases is explained by the large number of biotechnological applications. In this review, the most recent research on the overall structural features as well as on the structures and properties of the active sites are summarized, along with currently proposed mechanisms of reaction.
Keywords: laccase; dioxygen activation; copper