The antiproliferative effects of the haemoglobin beta-chain fragment (33-39) (valorphin or VV-haemorphin-5) were studied in a panel of tumour cell lines and normal cells of different origin, using various methods of activity determination (trypan blue inclusion test, sulphorhodamine B staining, MTT staining, flow cytometry and clonogenic test). Valorphin suppressed the proliferation of tumour cells by 25%-95%, depending on the cell line. The maximal valorphin activity was detected in transformed cells of fibroblastic (L929) and epithelial (MCF-7) origin, transformed haematopoietic cells (K562, HL-60) being less sensitive. In normal cells, valorphin activity was several fold lower (10%-15%). A study of the dynamics of cell proliferation in L929 cells using a visual cell count and flow cytometry showed that valorphin induced reversible and relatively short (24 h) S-phase arrest of cell proliferation, accompanied by a reversible increase of cell size. The proliferation delay was followed by a comparatively long period of reversible resistance of the cells to the peptide (96 h) when the cells are dividing at normal rate. The same dynamics were demonstrated for A549, MCF-7 and primary murine breast carcinoma cells. On the basis of the data obtained, a pattern of regulation of cell growth by valorphin is suggested.

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Streptococcus pyogenes (group A Streptococcus; GAS) is an important gram-positive extracellular bacterial pathogen responsible for a number of suppurative infections. This micro-organism has developed complex virulence mechanisms to avoid the host's defenses. We have previously reported that SDSC from GAS type M22 causes endothelial-cell dysfunction, and inhibits cell adhesion, migration, metabolism, and proliferation in a dose-dependent manner, without affecting cell viability. This work aimed to isolate and characterize a component from GAS type M22 supernatant that suppresses the proliferation of endothelial cells (EA.hy926). In the process of isolating a protein possessing antiproliferative activity we identified arginine deiminase (AD). Further study showed that this enzyme is most active at pH 6.8. Calculating Km and Vmax gave the values of 0.67 mmol·L(-1) and 42 s(-1), respectively. A distinctive feature of AD purified from GAS type M22 is that its optimum activity and the maximal rate of the catalytic process is close to neutral pH by comparison with enzymes from other micro-organisms. AD from GAS type M22 suppressed the proliferative activity of endothelial cells in a dose-dependent mode. At the same time, in the presence of AD, the proportion of cells in G0/G1 phase increased. When l-Arg was added at increasing concentrations to the culture medium containing AD (3 μg·mL(-1)), the enzyme's capacity to inhibit cell proliferation became partially depressed. The proportion of cells in phases S/G2 increased concomitantly, although the cells did not fully recover their proliferation activity. This suggests that AD from GAS type M22 has potential for the suppression of excessive cell proliferation.

Expression of gene of arginine deiminase (AD) allows adaptation of Streptococcus pyogenes to adverse environmental conditions. AD activity can lead to L‐arginine deficiency in the host cells’ microenvironment. Bioavailability of L‐arginine is an important factor regulating the functions of the immune cells in mammals. By introducing a mutation into S pyogenes M46‐16, we obtained a strain with inactivated arcA/sagp gene (M49‐16 delArcA), deficient in AD. This allowed elucidating the function of AD in pathogenesis of streptococcal infection. The virulence of the parental and mutant strains was examined in a murine model of subcutaneous streptococcal infection. L‐arginine concentration in the plasma of mice infected with S pyogenes M49‐16 delArcA remained unchanged in course of the entire experiment. At the same time mice infected with S pyogenes M49‐16 demonstrated gradual diminution of L‐arginine concentration in the blood plasma, which might be due to the activity of streptococcal AD. Mice infected with S pyogenes M49‐16 delArcA demonstrated less intensive bacterial growth in the primary foci and less pronounced bacterial dissemination as compared with animals infected with the parental strain S pyogenes M46‐16. Similarly, thymus involution, alterations in apoptosis, thymocyte subsets and Treg cells differentiation were less pronounced in mice infected with S pyogenes M49‐16 delArcA than in those infected with the parental strain. The results obtained showed that S pyogenes M49‐16 delArcA, unable to produce AD, had reduced virulence in comparison with the parental S pyogenes M49‐16 strain. AD is an important factor for the realization of the pathogenic potential of streptococci.