[Recombinant fragment of pigment epithelium-derived factor (44-77) prevents pathological corneal neovascularization].

Pigment epithelium-derived factor (PEDF), a 50 kDa secreted glycoprotein, is among the most potent endogenous inhibitors of angiogenesis. PEDF-derived fragment (44-77) possesses antiangiogenic properties of the full-sized protein and is a potential drug candidate for the treatment of ocular neovascular diseases. In this study we propose an efficient scalable biotechnological method for the production of PEDF (44-77) as part of a fusion protein with SspDnaB intein. The fusion protein was obtained in bacterial E. coli cells in the form of inclusion bodies, solubilized and subjected to autocatalytic cleavage with the release of PEDF (44-77) (yield, 77%). The target peptide was separated from the intein using tangential ultrafiltration. The final purification of PEDF (44-77) was performed by reversed-phase HPLC. The yield of the target peptide (purity, 99%) was 65 mg per 1 liter of culture. Antiangiogenic activity of the obtained peptide was studied in vitro using murine endothelial cells SVEC-4-10. PEDF (44-77) suppressed proliferation of endothelial cells by 53% and inhibited endothelial cell tube formation at the concentration of 1 nM. The ability of the recombinant PEDF (44-77) to block initial stages of angiogenesis was demonstrated using the model of rabbit corneal neovascularization.

[Recombinant oxyntomodulin].

An artificial gene encoding oxyntomodulin was obtained using chemical and enzymatic methods and cloned into Escherichia coli. A recombinant plasmid was constructed containing a hybrid oxyntomodulin gene and Ssp dnaB intein from Synechocystis sp. The expression of the resulting hybrid gene in E. coli, its properties, and the conditions of its autocatalytic cleavage to oxyntomodulin were studied.

[Recombinant thymosin alpha1]. / Rekombinantnyi timozin alpha1.

An artificial gene encoding thymosin alpha1 was obtained by the chemoenzymatic synthesis and cloned into Escherichia coli. An expressing recombinant plasmid containing the hybrid protein gene, which encodes amino acid sequences of thymosin alpha1 and the Saccharomyces cerevisiae intein Sce VMA, was constructed. The expression of the hybrid protein from the resulting hybrid gene in E. coli, the properties of the resulting hybrid protein, and the conditions for its nonenzymatic cleavage to thymosin alpha1 were studied. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 5; see also http: // www.maik.ru.

[An inhibitor of enteropeptidases and trypsin from the bovine duodenum]. / Ingibitor énteropeptidazy i tripsina iz dvenadtsatiperstnoi kishki byka.

Enteropeptidase inhibitor (DI) was isolated from bovine duodenum during purification of this enzyme. DI was purified by affinity chromatography on immobilised trypsin. DI preparations contain two main components: DI-9 (9 kD) and DI-20 (20 kD). The N-terminal amino acid sequence 1-19 of DI-9 is highly homologous to the Kunitz inhibitor (BPI). Molecular weights of DI-9 and BPI are the same (gel electrophoresis data). Fragment 1-19 of DI-9 differs from the corresponding region of BPI only at the position 17: DI-9 contains Ala-17 instead of Arg in BPI. The homology of N-terminal amino acid sequence 1-25 of DI-20 with the corresponding regions of some phospholipases A2 suggests that this protein is a new intestinal phospholipase A2. Inhibitor DI-9 and phospholipase DI-20 are probably isolated in a common lipoprotein complex. The only earlier known in vitro inhibitor of enteropeptidase, BPI, was localised in vivo in different tissues with this enzyme. In our opinion the Kunitz-type inhibitor DI-9 is, a physiological inhibitor of enteropeptidase.

[Genetic engineering of oxytocin: isolation of oxytocinoyllysine]. / Genno-inzhenernyi oksitotsin: vydelenie oksitotsinoillizina.

A hybrid protein, Il-Ox-K, was obtained from cells of E. coli TG1/pTOTEilox strain. The N-terminal sequence of this protein (63 amino acid residues) is a fragment of human interleukin-3, and the C-terminal sequence represents the full amino acid sequence of oxytocin flanked by a lysine residue. The modified oxytocinoyl-Lys containing S-sulfocysteine residues was isolated after tryptic digestion of S-sulfoderivative of the hybrid protein. The modified peptide was converted into the cyclic form containing the disulfide bonds [formula: see text]. Obtaining the oxytocinoyl-Lys proves the possibility of preparing short peptides using the microbiological synthesis.

[Effect of the antitumor antibiotic actinoxanthine on cells cultured in vitro]. / Vliianie protivoopukholevogo antibiotika aktinoksantina na kletki, kul'tiviruemye in vitrol

Significant changes in the nucleus structure, complete suppression of the mitotic activity, markedly decreased synthesis of RNA (by 70--80 per cent according to incorporation of 3H-uridine) and decreased levels of DNA (by 40 per cent according to olivomycin binding) were observed in the fibroblasts cultivated in vitro due to exposure to actinoxanthine in an amount of 50 microgram/ml. The data indicate direct damaging effect of the drug on the cell chromatin. The above nuclear changes were also observed after a short-term exposure of the cells to the drug (up to 5 minutes). Still, they became evident only after the subsequent incubation of the cells in a pure culture medium for at least 15 minutes. No such changes in the nucleus structure were detected when after the 5-minute exposure to actinoxanthine the cells were exposed to trypsin for 3 minutes. When the time of exposure to actinoxanthine was longer (15 minutes and higher), trypsin suppressed the manifestation of the above nuclear changes. The two-stage mechanism of the damaging effect of actinoxanthine on the chromatin of the cells cultivated in vitro is discussed. The damaging effect of actinoxanthine on the cells begins from binding of the drug with the cell membrane. After that a short incubation period follows and then the characteristic changes in the nucleus structure appear.

Chemical studies on actinoxanthin.

The antitumor protein actinoxanthin exhibits high inhibitory activity against a number of gram-positive bacteria and some strains of transplantable leucoses and related tumors. Actinoxanthin was shown to consist of a single polypeptide chain crosslinked by two disulfide bonds and to contain 107 amino acid residues. Reduced and alkylated actinoxanthin was digested with chymotrypsin, thermolysin and trypsin. Based on the sequence analysis of fragments so obtained the complete amino acid sequence and the location of disulfide bonds of actinoxanthin has been proposed. The high degree homology of some regions of actinoxanthin and the antitumor protein neocarzinostatin have been revealed.