[Fragments of functional proteins in the primary culture of human erythrocytes].

According to previously reported data, the supernatant of a primary culture of human erythrocytes contains 33 hemoglobin fragments. An analysis of the supernatant of a 20% (v/v) suspension of human erythrocytes allowed us to identify additionally four peptides whose precursors are cytoplasmic beta-actin (two fragments), fructose diphosphate aldolase B, and an unknown protein, as well as the amino acids tyrosine and tryptophan. The composition and the content of the components of the supernatant did not depend on the age or blood group of donors.

beta-Actin-derived peptides isolated from acidic extract of rat spleen suppress tumor cell growth.

Twenty-two fragments of beta-actin and beta-actin-related protein were isolated from the acidic extracts of rat spleen tissue. beta-Actin fragments (75-90), (78-89), and (78-88), 0.01-1 microM, decreased live cell number of L929 murine tumor fibroblasts by 80-90%, with maximal cytotoxic effect of 30-40%.

Stimulation of fibroblast proliferation by neokyotorphin requires Ca influx and activation of PKA, CaMK II and MAPK/ERK.

Neokyotorphin [TSKYR, hemoglobin alpha-chain fragment (137-141)] has previously been shown to enhance fibroblast proliferation, its effect depending on cell density and serum level. Here we show the dependence of the effect of neokyotorphin on cell type and its correlation with the effect of protein kinase A (PKA) activator 8-Br-cAMP, but not the PKC activator 4beta-phorbol 12-myristate, 13-acetate (PMA). In L929 fibroblasts, the proliferative effect of neokyotorphin was suppressed by the Ca2+ L-type channel inhibitors verapamil or nifedipine, the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester, kinase inhibitors H-89 (PKA), KN-62 (Ca2+/calmodulin-dependent kinase II) and PD98059 (mitogen-activated protein kinase).

Antitumor effect of valorphin in vitro and in vivo: combined action with cytostatic drugs.

The action of the cytostatic drugs (epirubicin and vincristine) in combination with the endogenous antiproliferative beta-hemoglobin fragment (33-39), valorphin, was studied in tumor (L929 and A549) cell cultures, primary culture of murine bone marrow cells and in murine model of breast carcinoma in vivo. Simultaneous application of 1 microM valorphin and 1 microM epirubicin, in vitro, did not result in an additive suppressive effect on cell culture growth. Additive effects were achieved with alternating applications of the peptide and the drugs, namely, 0.

What to synthesize? From Emil Fischer to peptidomics.

The driving forces, incentives and strategic targets of peptide synthesis have undergone considerable evolution during the centenary following the pioneer work of Emil Fischer. In those days peptide synthesis was considered as a way of confirming the polypeptide theory of protein structure. The scientific community also expected (naively) that the synthesis would eventually lead to the creation of artificial living organisms.

Proliferative activity of neokyotorphin-related hemoglobin fragments in cell cultures.

alpha-Hemoglobin fragments alpha-(133-141), alpha-(134-141), alpha-(135-141), alpha-(137-141), alpha-(134-140), alpha-(133-138), alpha-(134-140) and alpha-(137-138) stimulate L929 tumor cell proliferation, alpha-(134-141) being the most active. alpha-(134-141) stimulates proliferation of M3 melanoma cells, murine embryonic fibroblasts, primary cultures of red bone marrow and spleen cells. In L929 cells the effect of alpha-(134-141) is cell density independent; in M3 cells alpha-(137-141) and alpha-(134-141) are most active at density 10,000 cells/well (96 well plate) independently on FBS content.

Antiproliferative action of valorphin in cell cultures.

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.

Family of hemorphins: co-relations between amino acid sequences and effects in cell cultures.

Hemorphins, i.e. endogenous fragments of beta-globin chain segment (32-41) LVVYPWTQRY(F) suppress the growth of transformed murine fibroblasts L929 cell culture, the effect is due to cytotoxicity and inhibition of cell proliferation.

Endogenous fragment of hemoglobin, neokyotorphin, as cell growth factor.

It is shown that neokyotorphin (the alpha-globin fragment 137-141) stimulates proliferation of normal cells (murine embryonic fibroblasts, red bone marrow and spleen cells) and tumor cells (murine melanoma and transformed fibroblasts L929) in the absence or in the presence of fetal bovine serum. In contrast to serum deprivation conditions, the ability to potentiate L929 cell growth in the presence of fetal serum is strongly cell density dependent. The peptide also enhances the viability of L929 cells, murine embryonic fibroblasts and of the primary cultures of murine red bone marrow cells and splenocytes under serum-deprivation conditions for at least 72 h.

Peptides comprising the bulk of rat brain extracts: isolation, amino acid sequences and biological activity.

Chromatographic separation of rat brain extracts followed by automatic Edman sequencing of the major individual components resulted in identification of 61 endogenous peptides derived from known functional proteins (hemoglobin, myelin basic protein, cytochrome-c oxidase, etc.) or unknown precursors. The results are compared with the data obtained earlier for bovine brain.

[Proteolytic degradation of hemoglobin in erythrocytes results in formation of biologically active peptides].

The formation of biologically active hemoglobin fragments in human erythrocytes was studied. The structures of 33 peptide products of intraerythrocytic hemoglobin cleavage were determined. Based on an analysis of these sequences, a model of the stepwise degradation of the hemoglobin alpha- and beta-chains was suggested.

Neokyotorphin and neokyotorphin (1-4): secretion by erythrocytes and regulation of tumor cell growth.

Human erythrocytes release neokyotorphin, the 137-141 fragment of hemoglobin alpha-chain into the supernatant of red blood cells primary culture. However, the neokyotorphin fragment 1-4 that is formed together with neokyotorphin inside the red blood cells and in various tissues is not found in the supernatant. Both neokyotorphin and its 1-4 fragment were shown to stimulate proliferation of L929 tumor cells.

Cytotoxic activity of acetylcholine receptor ligands.

Acetylcholine receptor ligands were studied for cytotoxicity in K562 human erythroid leukemia tumor cells. Cytotoxicity of carbachol, an agonist of acetylcholine receptors, atropine, an antagonist of muscarinic acetylcholine receptor, neurotoxin 11 (NT II) from Naja naja oxiana cobra venom and tubocurarine, antagonists of acetylcholine receptor of nicotinic type was exhibited in the 10-7-10-5 M concentration range. Several cytolytic processes, two for carbachol and three for other ligands, corresponding to different concentrations of each ligand were detected.

Met-enkephalin induces cytolytic processes of apoptotic type in K562 human erythroid leukemia cells.

Cytolytic activity of Met-enkephalin, an endogenous opioid peptide, was studied within the 10(-7)-10(-17) M concentration range in K562 human erythroid leukemia cells. Cytolytic activity was determined by the trypan blue inclusion method after 13, 15 and 18 h of Met-enkephalin co-incubation with target cells. Discrete maxima of cytolytic activity were detected at concentrations of 10(-9)-10(-10), 10(-13) and 10(-15) M.