Specific Cytotoxicity of Targeted 177Lu and 212Pb-Based Radiopharmaceuticals.

Two radiopharmaceutical preparations were developed on the basis of artificial targeted polypeptide ZHER2 specific to HER2/neu tumor marker and radionuclides 177Lu (ZHER2-HSA-chelator-177Lu) or 212Pb (ZHER2-HSA-chelator-212Pb). The objective was to evaluate in vitro the cytotoxic activity of the targeted radiopharmaceuticals using two cultured human breast cancer cell lines with different expression of HER2/neu: SK-BR3 (high expression of HER2/neu) and MCF-7 (low expression of HER2/neu). It was shown that the cytotoxic effect of both preparations was significantly higher against the SK-BR-3 cells. The cytotoxicity correlated with the incubation period (it was higher after 72 h than after 24 h) and was significantly more pronounced in comparison with activity of radionuclide salts without a specific ligand. In vivo preclinical study of these pharmaceuticals seems to be very promising in animals with xenografted tumors showing high expression of HER2/neu marker.

[Biosynthesis and purification of human beta2-adrenergic receptor expressed in methylotrophic yeast Pichia pastoris].

Human beta2-adrenergic receptor is one of the most studied G-protein-coupled receptors. It plays a key role in autonomic nervous system and is a drug target in cardiovascular and pulmonary diseases. Despite the fact that its crystal structure was revealed, a physiological role and molecular mechanisms of its action remain largely unknown. We designed the construct pVR2ADRH, which contained the gene for human beta2-adrenergic receptor with a polyhistidine tag C-terminal extension. The recombinant DNA was used for transformation of the GS115 strain of Pichia pastoris. The heterologous expression level obtained was about 20 mg/l. The receptor was extracted from membrane fraction and was purified by metal-affinity and ion-exchange chromatography. The active receptors were isolated by alprenolol-sepharose CL-4B. The resulting level of purified human beta2-adrenergic receptor was approximately 1 mg per liter of culture. The homogeneity of the protein sample was confirmed by a dynamical light scattering analysis of the receptor's micellar solution.

[Bacterial synthesis, purification, and solubilization of transmembrane segments of ErbB family members].

A family of epidermal growth factor receptors, ErbB, represents an important class of receptor tyrosine kinases, playing a leading role in cellular growth, development and differentiation. Transmembrane domains of these receptors transduce biochemical signals across plasma membrane via lateral homo- and heterodimerization. Relatively small size of complexes of ErbB transmembrane domains with detergents or lipids allows one to study their detailed spatial structure using three-dimensional heteronuclear high-resolution NMR spectroscopy. Here, we describe the effective expression system and purification procedure for preparative-scale production of transmembrane peptides from four representatives of ErbB family, ErbB1, ErbB2, ErbB3, ErbB4, for structural studies. The recombinant peptides were produced in Escherichia coli BL21(DE3)pLysS as C-terminal extensions of thioredoxin A. The fusion protein cleavage was accomplished with the light subunit of human enterokinase. Several (10-30) milligrams of purified isotope-labeled transmembrane peptides were isolated with the use of a simple and convenient procedure, which consists of consecutive steps of immobilized metal affinity chromatography and cation-exchange chromatography. The purified peptides were reconstituted in lipid/detergent environment (micelles or bicelles) and characterized using dynamic light scattering, CD and NMR spectroscopy. The data obtained indicate that the purified ErbB transmembrane peptides are suitable for structural and dynamic studies of their homo- and heterodimer complexes using high resolution NMR spectroscopy.

[X-ray diffraction and biochemical studies of W34F mutant ribonuclease binase].

The structures of two crystal modifications of the W34F mutant ribonuclease from the bacterium Bacillus intermedius (binase) were solved and refined at 1.7 and 1.1 A resolution. The kinetic parameters of the hydrolysis of substrates of different lengths (GpU, GpUp, and poly(I)) by binase and its W34F mutant were investigated and compared. The catalytic activity of the enzymes was shown to increase with increasing length of the substrate. The substitution of tryptophan for phenylalanine does not lead to a change in the activity of the enzyme but results in a decrease in the binding constants for substrates containing more than one phosphate groups. A comparison of the structure of the mutant enzyme with the previously established structures of binase and its complexes with sulfate ions and guanosine monophosphate showed that the difference in their kinetic parameters is related to the fact that the mutant ribonuclease cannot bind the second phosphate group. Both crystal modifications of the mutant binase contain dimers, like in the crystal structure of binase studied previously. In these dimers, only one enzyme molecule can bind the substrate molecule. Since the dimers were found in the crystals grown under four different conditions, it can be suggested that the enzyme can exist as dimers in solution as well. Mutants of binase, which could exclude the formation of dimers, are suggested.

[Residual dipolar couplings and molecular dynamic calculations as a source for refinement of protein spatial structures].

The precision of techniques and factors affecting the interpretation of residual dipolar couplings (RDCs) in analysis of spatial structures of partially aligned proteins are discussed. Experimental RDC values were obtained for pairs of 1H-15N nuclei of the protein barstar partially aligned in a liquid crystalline matrix of bicelles composed of dimiristoylphosphatidylcholine and dihexanoylphosphatidylcholine. The observed couplings agree well with the spatial structures of barstar determined earlier by X-ray and NMR methods. However, the differences between the experimental and calculated RDCs that were calculated on the basis of the known spatial structures of barstar, exceed the experimental errors three- to fourfold. These discrepancies can be explained by differences in the protein structures in solution and in crystal, a limited precision of the X-ray analysis, and the intramolecular mobility of the protein molecule. A comparison of the results of modeling of the molecular dynamics of barstar in solution, crystal structures, and the experimental RDCs showed that the methods of molecular dynamics provide for a reasonable description of the character and amplitudes of internal motions and they should be considered for the correct determination of protein spatial structures from NMR spectroscopic data.

Thin-film conductometric biosensors for glucose and urea determination.

The characteristics of the developed conductometric biosensors for urea and glucose determination are described. Conductometric transducers based on thin-film interdigitated metal (Au, Cr, Cu, Ni) electrodes were studied, and enzymes urease and glucose oxidase were used for the selective membranes formation on the chips having gold electrodes. The influence of ionic strength and buffer capacity of the samples on the biosensors response in kinetic and steady-state modes of measurements was thoroughly tested. It was shown that the kinetic response of the sensors does not depend on the buffer capacity of the analyzed sample. In basic features the performance of the developed biosensors is rather close to that of respective enzyme field effect transistor, though the former are much superior when the technological complexity of the transducer itself is considered and taking into account that conductometric sensors require no reference electrode.

[Theoretical analysis of the amino acid sequence of receptors for growth hormones and prolactins. Prediction of ligand-binding segments]. / Teoreticheskii analiz aminokislotnykh posledovatel'nostei retseptorovgormonov rosta i prolaktinov. Predskazanie ligand-sviazyvaiushchikh uchastkov.

At present the growth hormone and prolactin receptors were cloned along with their variant forms from human, rat, mouse, rabbit, bovine and sheep tissues. The functional topography of receptors is practically unknown. Because of the high price and difficulty of protein's total mutagenesis, it is reasonable to carry on a theoretical analysis of structures of receptors to predict the most probable ligand-binding sites. We studied the primary structures of known prolactin and growth hormone receptors using theoretical methods proved to be powerful in earlier structure--activity relationship investigations. We analyzed the secondary structure, conservative positions, hydrophilicity profiles of the growth hormone and prolactin receptors, and used the original method based on information theory to predict the sites which are promising for mutagenesis or peptide synthesis as probable ligand-binding sites. Three segments corresponding to the main conservative, hydrophilic and rare sites were predicted to form the ligand-binding determinant.

[Superproduction of Bacillus intermedius 7P ribonuclease (binase) in Escherichia coli]. / Superproduktsiia ribonukleazy Bacillus intermedius 7P (binzay) v Escherichia coli.

We have reported previously about the cloning of the binase gene in E. coli. In this work, using an original approach named "homolog gene recombination" method (HGR), vectors for binase expression in E. coli have been constructed. Transcription of the binase gene have been directed through either tac-promoter or PR-promoter of bacteriophage lambda under the control of temperature-sensitive CI857 repressor. The last promoter gave the maximum yield of binase, up to 100 mg of protein per litre of heat-induced bacterial culture. The location of the transcription terminator at the 3' terminus of the binase gene raised the expression approximately two times more. A chromatographic method have been developed and applied for the control of binase accumulation in growth medium without measuring the ribonuclease activity.

[Cloning of the gene for extracellular Bacillus circulans RNAase]. / Klonirovanie gena vnekletochnoi RNKazy Bacillus circulans.

The gene for extracellular low molecular weight ribonuclease of Bacillus circulans BCF 247 was cloned. The strain was isolated from permafrost deposits of the Kolyma lowland. The gene for the ribonuclease from Bacillus intermedius (binase) was used as a specific probe. The cloning succeeded only in the E. coli strain producing the inhibitor of ribonuclease form Bacillus amyloliquefaciens. Selected clones secreted the active ribonuclease into the growth media. Deletion derivatives of the parental recombinant plasmid were constructed. The smallest DNA fragment which enclosed a functional ribonuclease gene in E. coli was determined to be 0.6 kb in length.

[Ribonuclease from Bacillus thuringiensis var. subtoxicus. Gene structure and biosynthesis regulation]. / Ribonucleaza iz Bacillus thuringiensis var. subtoxicus. Struktura gena i reguliatsiia biosinteza.

The gene for extracellular guanyl-specific ribonuclease of Bacillus thuringiensis var. subtoxicus (RNase Bth), a close homologue of the B. intermedius RNase (binase), was completely sequenced. Analysis of nucleotide sequences in the regions adjoining RNase genes revealed an identical organization of the chromosomal loci of RNase Bth and binase. Growth characteristics of the Bacillus thuringiensis var. subtoxicus strain and its synthesis of RNase were studied. It was shown that the exogenous inorganic phosphate inhibits the biosynthesis of RNase. At the same time, actinomycin D in low doses stimulates the enzyme synthesis. Comparative analysis of the influence of inorganic phosphate and actinomycin D on the biosynthesis of RNAse Bth and binase suggests a possibility of coincidence of regulatory pathways of synthesis of these enzymes.

[Expression of neurotoxin II from Naja oxiana cobra venom in Escherichia coli in a hybrid form with thioredoxin]. / Ekspressiruiushchaia konstruktsiia dlia narobotki v Escherichia coli neirotoksina i iz iada kobry Naja oxiana v vide gibrida s tioredoksinom.

Neurotoxin II from the venom of cobra Naja oxiana is a short type alpha-neurotoxin, which competitively inhibits nicotinic acetylcholine receptor. The toxin gene was expressed as a construct fused with the thioredoxin gene and the linker encoding the enteropeptidase recognition site and a Met residue between the genes. The fusion protein was mainly cleaved by cyanogen bromide, since enteropeptidase was less effective. The yield of neurotoxin II was 6 mg/l of the bacterial culture. The resulting recombinant protein was identified with native neurotoxin II by its N-terminal analysis, mass spectrometry, and NMR spectroscopy. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 1; see also http://www.maik.ru.

[Secondary structure of binase in solution by 1H NMR]. / Vtorichnaya structura binazy v rastvore po dannym 1H-YaMR spectroskopii.

Nearly all resonances were assigned in the two-dimensional 1H NMR spectra of binase, guanylospecific ribonuclease from Bacillus intermedius containing 109 amino acid residues. The exchange rates of amide protons with the solvent deuterium were measured in 2H2O at pH 6.7 and 30 degrees C. Coupling constants 3J of H-NC alpha-H, NOE contacts, solvent exchange rates of amide protons, and indices of C alpha H chemical shifts were measured, and the binase secondary structure was deduced from these data. It involves three alpha-helices in the N-terminal part (the 6-16, 26-31, and 41-45 segments) and a beta-sheet formed by five antiparallel beta-strands (51-55, 71-75, 86-90, 95-99, and 104-108 segments). The binase secondary structure was compared with that of its closest homologue, barnase from B. amyloliquefaciens.

[The study of the metal-binding region in human growth hormone using immobilized metal ion affinity gel-electrophoresis]. / Izuchenie metallosviazyvaiushchego uchastka v gormone rosta cheloveka metodom metall-khelatnogo affinnogo gel'-élektroforeza.

The zinc(II)-binding affinities of recombinant human growth hormone and two its mutants, 14-33 and 14-95, were studied using Immobilized Metal Ion Affinity Gel-electrophoresis (IMAG). The mutant hormones, composed of polypeptide chain segments of the human and porcine growth hormones, lacked His18, which may be crucial for binding of the intact hormone to the transition metal ions. The mutations did not affect the affinity of human growth hormone to immobilized zinc ions; the structural analysis implied that the human growth hormone contains two IDA-Zn(II) potential sorption sites formed by amino acid residues His21, Asp171, and Glu174 and/or His18 and Glu174.

[I87E mutation prevents barstar dimerization]. / Mutatsiia I87E prepiatstvuet dimerizatsii barstara.

The C40,82A;I87E mutant of barstar, an intracellular inhibitor of the ribonuclease barnase from Bacillus amyloliquefaciens, was obtained, and its physicochemical properties were studied. It was produced as a fusion protein with thioredoxin and then cleaved from this by EKmax enterokinase. The mutant was shown by NMR to retain the spatial structure of the wild-type protein but, in contrast to barstar, does not form the homodimers characteristic of barstar in aqueous solution. The mutant protein binds barnase with the dissociation constant (6.6 +/- 1.1) x 10(-11) M and exhibits other physicochemical properties similar to those of the wild-type barstar. This allows the use of C40,82A;I87E mutant instead of wild-type barstar in investigations where the protein dimerization is undesirable. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 6; see also http://www.maik.ru.

[Use of conductometric microsensors for studying kinetic parameters of enzymes]. / Ispol'zovanie konduktometricheskikh mikrosensorov dlia izucheniia kineticheskikh parametrov fermentov.

An alternative method based on thin-film conductometric microsensor is suggested for studying enzyme kinetics. It is established that the immobilized enzyme as well as soluble one is described by the classic lows of enzyme kinetics. Major parameters (Km and Vmax) are identical to those measured by the widely used methods and their amounts are 1.33 mM and 9.04 microS/min for soluble urease and 3.73 mM and 13.8 microS/min for immobilized urease; 5.26 mM and 13.9 microS/min for immobilized glucose oxidase; 13.6 mM and 122.0 microS/min for immobilized acetyl cholinesterase; 10.8 mM and 129.9 microS/min for immobilized butyryl cholinesterase. The possibility of conductometric analyzer application for detection of toxin concentration, in particular pesticides, is shown. Advantages and disadvantages of the system suggested are discussed.

[Amperometric enzyme biosensor with a glucose oxidase-polyaniline membrane]. / Amperometricheskii fermentnyi biosensor s membranoi gliukozooksidaza-polianilin.

An amperometric glucose biosensor was made by electrochemical polymerization of aniline onto the gold electrodes in presence of the enzyme glucose oxidase in the phosphate buffer solution with pH 7.0. Aniline is easily polymerized forming a thin film, which adheres tightly on the electrodes surface. During the electropolymerization process glucose oxidase was entrapped into polyaniline film which then became the catalyst of the enzyme reaction of glucose hydrolysis. Experiments were performed to determine optimal conditions of polyaniline-glucose oxidase film preparation. Glucose was amperometrically determined with the electrochemically fabricated biosensor in the concentration range 10(-4) M to 2 x 10(-2) M. The linearity of the enzyme electrode response ranged from 2 x 10(-4) M to 6 x 10(-3) M. The electrochemical synthesis of a polyaniline-enzyme thin film a high-technologic one and this permits fabricating various microbiosensors and multisensors in the continuous technological cycle.

[Optimal conditions for the functioning of a urease-based biosensor and pH-sensitive field transistors. Determination of urea in solution]. / Izuchenie optimal'nykh uslovii raboty biosensora na osnove ureazy i pH-chuvstvitel'nykh polevykh tranzistorov. Opredelenie mocheviny v rastvore.

The article deals with the optimal conditions of urea determination in solution by the enzyme sensor based on pH-sensitive field effect transistor. The dependence of response level on temperature, pH, ionic strength, buffer capacity were investigated. 10 mM phosphate buffer, pH 7.4 containing 200 mM NaCl is optimal for possible measurements of urea in diluted blood. Temperature change in the measured samples from 15 degrees to 30 degrees C does not affect the sensor signal value. Stability of immobilized ureas was investigated at storage under different conditions. In the presence of 1 mM EDTA activity of immobilized enzyme does not change during one month storage at 4 degrees C.