Nitroxide malonate methanofullerene as biomimetic model of interaction of nitroxide species with antioxidants.

Bis-nitroxide malonate methanofullerene (NO)2-MF was studied as a biomimetic model of reduction-oxidation activity with natural compounds-cytochrome c (cyt c), dihydroquercetin (DHQ), ascorbic acid (AA) and synthetic drug-1-(β-oxyethyl)-4,6-dimethyl-1,2-dihydro-2-oxopyrimidine (xymedon(®)). (NO)2-MF may be used as the component of Langmuir monolayers on an aqueous subphase and as the adsorbate on silica gel. The activity of (NO)2-MF in the reaction with cyt c was compared with the effect of nitroxide species such as gaseous nitric oxide, 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO) by using UV-vis and EPR-spectra.

The study of the complexes of nitromedicine with cytochrome c and NO-containing aqueous dosage form in the wound treatment of rats.

The interaction of cytochrome c with nitromedicines, such as 5-nitrofural, 5-nitroxoline, metronidazole and sodium nitrite which enables the generation of nitric oxide or nitrosyl complexes in the presence of ascorbic acid or sodium ascorbate in acid medium has been investigated. The pharmaceutical compositions containing cytochrome c and nitromedicine complexes as active substances were studied in the experiments by using rats. It has been shown that positive local and systemic effects were estimated when NO-containing gel was used at burn treatment.

[The influence of tumor necrosis factor alpha on the processes of sphingomyelin cycle and lipid peroxidation in brain].

The influence of tumor necrosis factor alpha (TNF-alpha) on the processes of sphingomyelin cycle activation and intensity of peroxidation in animal brain in vivo has been studied. Alterations in activity of sphingomyelinase, a key sphingomyelin cycle enzyme and in sphingomyelin, ceramide content as well as accumulation of the products of lipid peroxidation (diene conjugates and diene ketons) were measured in the cortex, the cerebellum and the hippocampus of rats 5, 15, 30 min, 1, 2 and 5 hours after TNF-alpha intraperitoneal injection in dosage 100 mkg per animal. It is shown that 2 hours after the injection, TNF-alpha initiated an accumulation of the products of lipid peroxidation, which intensively developed in the cerebellum and the hippocampus.

[Design of chimeric proteins based on a pentameric superhelical fragment of COMP. II. Properties of a hybrid, containing an VNTR (MUC1) antigen of human tumors].

An oligomeric chimeric protein DB-2 was constructed, to design drugs with antitumor activity and to develop highly sensitive immunospecific tests for the diagnostics of a wide variety of malignant epithelial cells in humans. DB-2 contains an immunodominant site of tetanus toxin and a fragment of the locus of the human tumor-associated antigen MUC1 with a variable number of tandem repeats. A pentameric superhelical fragment of the human cartilage oligomeric matrix protein was used as an oligomerization matrix.

[Design of chimeric proteins based on pentameric superhelical fragments of COMP. I. Properties of a hybrid containing an immunodominant segment of the surface protein of Plasmodium falciparum].

The oligomeric recombinant protein DB-1 containing the immunodominant sites of the circumsporozoite protein of Plasmodium falciparum and tetanus toxin was constructed to optimize the schemes of presentation of B-cell epitopes during vaccination with chimeric proteins without the use of adjuvants. A fragment of the pentameric coiled-coil human cartilage oligomeric matrix protein was used as an oligomerization matrix. The expression of the protein in Escherichia coli cells was studied, a method for its purification was developed, and it was biochemically characterized.

Immunodetection of Murine Lymphotoxins in Eukaryotic Cells.

Lymphotoxins alpha and beta (LTalpha and LTbeta) are members of tumor necrosis factor superfamily. LT heterotrimers exist on the surface of lymphocytes and signal through LTbeta receptor while soluble LTalpha homotrimer can signal through TNF receptors p55 and p75. LT-, as well as TNF-mediated signaling are important for the organogenesis and maintenance of microarchitecture of secondary lymphoid organs in mice and has been implicated in the mechanism of certain inflammatory syndromes in humans.

Role of tumor necrosis factor alpha and sphingomyelin cycle activation in the induction of apoptosis by ischemia/reperfusion of the liver.

The signal transduction pathways triggering apoptotic mechanisms after ischemia/reperfusion may involve TNF-alpha secretion, ceramide generation, and initiation of lipid peroxidation. In the present study involvement of the TNF-alpha, sphingomyelin cycle, and lipid peroxidation in the initiation of apoptosis induced in liver cells by ischemia and reperfusion was investigated. Wistar rats were subjected to total liver ischemia (for 15, 30 min, and 1 h) followed by subsequent reperfusion.

[Deletion mutants of human granulocyte-macrophage colony-stimulating factor].

To study the structure-function relationship of the human granulocyte-macrophage colony-stimulating factor (GM-CSF), genes were constructed that encode its three deletion mutants: D1, a mutant with the deletion of six amino acid residues (37-42) some of which are a part of a beta-structural region; D2, a mutant with the deletion of the unstructured six-aa sequence of a loop (45-50); and D3, a mutant with the deletion of 14 aa residues (37-50) corresponding to the A-B loop and encoded by the second exon of the gmcsf gene. The expression products of these genes in E. coli were accumulated in a fraction of insoluble proteins.

[Modified oligonucleotides containing 1-beta-D-galactopyranosylthymine: synthesis and substrate properties].

A convenient method of regioselective introduction of 1-beta-D-galactopyranosylthymine into oligonucleotides was developed and the substrate properties of the modified oligonucleotides were investigated in the enzymic reaction of formation and hydrolysis of internucleotide bonds. The English version of the paper.

Methods for preparation of recombinant cytokine proteins V. mutant analogues of human interferon-gamma with higher stability and activity.

Mutant analogues of recombinant human immune interferon (IFN-gamma) with higher stability and biological activity were prepared. Depending on the analogue, protein structure modification might involve introduction of an intramonomer disulfide bond (through replacements of Glu7Cys and Ser69Cys), C-terminal shortening by 10 amino acid residues, as well as Gln133Leu substitution in truncated variant. Isolation, purification, and renaturation of the IFN-gamma analogues expressed in Escherichia coli as inclusion bodies were performed according to the scheme developed earlier for wild-type protein.

[Chaperone Caf1M stabilizes hybrid proteins containing sequences of F1 antigen subunit from Yersinia pestis].

The Yersinia pestis (causative agent of plague) capsule antigen is a homopolymer of Caf1 protein. Export of the subunits is mediated by the periplasmic chaperone Caf1M. To study the mechanism of Caf1M activity, two hybrid genes including coding sequences for the Caf1 signal peptide, human granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-1 (IL-1) receptor antagonist, and mature Caf1 were constructed and expressed in Escherichia coli.

[Efficacy and perspective of application of preparation based on hydrogel and xerogel of methyl-silicic acid in patients with the intestinal malignancy].

There was studied the influence of medicinal preparations, created on the base of methylsiliconorganic matrix, on immediate and late follow-up result of treatment of more than 2000 patients with the digestive channel malignancy. High efficacy of application in complex of preoperative preparation of immobilized antibiotics and cytostatics, enterosorpent enterosgel for the purulent-inflammatory complications prophylaxis, lowering of bilirubin level in tumoral obturative jaundice, and in intraoperative usage for the tumor and metastases recurrences prophylaxis.

Secretion of recombinant proteins via the chaperone/usher pathway in Escherichia coli.

F1 antigen (Caf1) of Yersinia pestis is assembled via the Caf1M chaperone/Caf1A usher pathway. We investigated the ability of this assembly system to facilitate secretion of full-length heterologous proteins fused to the Caf1 subunit in Escherichia coli. Despite correct processing of a chimeric protein composed of a modified Caf1 signal peptide, mature human interleukin-1beta (hIL-1beta), and mature Caf1, the processed product (hIL-1beta:Caf1) remained insoluble.

[The ways of the efficacy raising in surgical treatment of patients with colonic cancer].

The indications extension for the radical surgical intervention conduction in elderly and senile patients and with locally spreading colonic cancer had promoted the result of their treatment improvement.

Recombinant human insulin. VIII. Isolation of fusion protein--S-sulfonate, biotechnological precursor of human insulin, from the biomass of transformed Escherichia coli cells.

Various methods have been investigated for the isolation and purification of fusion proteins of precursors of human insulin in the form of S-sulfonates, from the biomass of transformed Escherichia coli cells. Fusion proteins were prepared with different sizes and structures of the leader peptide and the poly-His position (inserted for purification by metal chelate affinity chromatography). The fusion proteins contained an IgG-binding B domain of protein A from Staphylococcus aureus at the N-terminus and an Arg residue between the leader peptide of the molecule and the proinsulin sequence, for trypsin cleavage of the leader peptide.

[Immunoenzyme determination of human granulocyte-macrophage colony-stimulating factor using monoclonal antibodies].

A set of seven hybridomas producing monoclonal antibodies (MAbs) to the human recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) was obtained. The properties of the monoclonal antibodies were characterized, and pairs of MAbs specific to different non-overlapping epitopes of GM-CSF were identified. A sensitive and simple method of two-site ELISA for GM-CSF was developed on the basis of two MAbs.

[Primary multiple malignant tumors of the stomach and colon].

Experience of diagnosis and treatment of primarily-multiple gastric cancer, combined with malignant colonic tumors in 27 patients was summarized. One-stage combined radical treatment of the patients is the most effective one.

[Heterologous expression of murine lymphotoxins in Escherichia coli and preparation of antibodies].

Genes encoding fragments of polypeptide chains of murine lymphotoxins (LT), namely, LT-alpha truncated from the N-terminus and the LT-beta extracellular domain, containing N-terminal hepta- and hexahistidine epitopes, respectively, were expressed in E. coli cells. The recombinant proteins purified by metallochelate chromatography were used to obtain polyclonal antibodies that specifically recognize murine LT.

TNF-induced haptoglobin release from human neutrophils: pivotal role of the TNF p55 receptor.

Haptoglobin (Hp), TNF-alpha, and neutrophils are parts of a highly interactive ensemble participating in inflammatory processes. Hp is taken up by neutrophils, stored within a cytoplasmic granular compartment, and is secreted during phagocytosis by those cells. In the present study, the effects of TNF-alpha on the release of Hp from human neutrophils were investigated.

Tumor necrosis factor mutants with selective cytotoxic activity.

Tumor necrosis factor (TNF-alpha) has a cytotoxic or cytostatic effect when tested with various malignant cell lines. Clinical trials in cancer patients, however, revealed high systemic toxicity of TNF-alpha. The existence of two types of receptor may partially explain the pleiotropic activity of TNF-alpha.

Methods of preparation of recombinant cytokine proteins.

Two schemes for efficient and productive isolation for mutant human recombinant tumor necrosis factor-alpha (TNF-alpha R32H) from Escherichia coli cells were developed. The methods include membrane filtration, ion-exchange chromatography and gel filtration, and centrifugation with subsequent free-flow electrophoresis as an alternative procedure. The target product was obtained as active trimer with total yield more than 50% and greater than 98% purity according to PAGE, size-exclusion chromatography, HPLC, and HPCE.

Relation of biological activity of mutant forms of recombinant human tumor necrosis factor alpha and accumulation of sphingosine in murine liver.

TNF-alpha induced sphingomyelin hydrolysis by sphingomyelinase and both sphingosine and ceramide generation have been reported to be implicated in a number of TNF-alpha responses, including cytotoxicity and apoptosis. We found that sphingosine, a highly cytotoxic product of enzymatic degradation of sphingomyelin, is accumulated in liver of mice treated with TNF-alpha. To determine the role of sphingosine in TNF-alpha toxicity, TNF-alpha mutants differing in their cytotoxicity to L929 cells as well as haemorrhagic tumor necrosis, tumor regression and lethal toxicity in mice were used in our experiments.

[Interaction of anti-TNF-alpha monoclonal antibodies E7H2 with mutant and hybrid proteins].

A number of hybrid proteins containing amino acid sequences of human lymphotoxin and human and mouse tumor necrosis factors were obtained by means of genetic engineering. By using these proteins, an antigenic determinant in the molecule of human tumor necrosis factor for monoclonal antibodies E7H2 (MAb E7H2), previously developed against this factor was localized. It was demonstrated by Western blot analysis that the MAb E7H2-binding site is located in the sequence region 37-49 of human tumor necrosis factor and includes the sequence Val41GluLeuArg44 directly interacting with MAb E7H2.