The antioxidant effect of fermented papaya preparation involves iron chelation.

Iron-overload is a major clinical problem in various diseases. Under this condition, serum iron which surpasses the binding capacity of transferrin is present as non-transferrin bound iron and cellular unbound Labile Iron Pool (LIP) is increased. LIP participates in the generation of free radicals, including reactive oxygen species (ROS). Increased ROS, with concomitant decrease in anti-oxidants, results in oxidative stress and toxicity to the liver, heart and other tissues, causing serious morbidity and eventually mortality. Therapeutic iron chelation reduces the LIP and thereby ameliorates oxidative stress-mediated toxicity. Many food-derived antioxidants have the capacities to scavenge ROS and chelate iron. We have reported that fermented papaya preparation (FPP) has ROS scavenging effect on blood cells in vitro or in vivo (in thalassemic patients and experimental animals). We now investigated FPP's iron chelating effect - its ability to prevent (and revert) LIP accumulation. Liver- and heart-derived cells, and RBCs were exposed to non-transferrin bound iron in the form of ferrous ammonium sulfate and the effect of FPP on their LIP content and ROS generation was measured by flow-cytometry. The results indicate that FPP reduces LIP and ROS, and suggests that its antioxidant mechanism is related, at least in part, to iron chelation.

Differentiation of human erythroid cells in culture.

A culture procedure for growing erythroid progenitors in liquid medium is described. The procedure is divided into two phases. The first is an erythropoietin (EPO)--independent phase in which peripheral blood mononuclear cells are isolated and cultured in the presence of a combination of growth factors, but in the absence of EPO. During this phase, early erythroid-committed progenitors proliferate and differentiate into more mature progenitors. In the second phase, the latter cells, cultured in an EPO-supplemented medium, continue to proliferate and mature into hemoglobin-containing nucleated erythroid cells. The culture procedure yields populations that are large, relatively pure, and synchronized (in terms of differentiation), and which recapitulate in vivo erythropoiesis. Since the cells are grown in suspension, samples of cells can be withdrawn at any time, without disturbing the cultures, and assayed for various parameters, e.g., morphology, size, number, viability, apoptosis, cell cycle, surface antigens, or gene expression. Several procedures for analyzing the cultured cells with respect to their hemoglobin content during their differentiation are included.

Prodrugs of butyric acid. Novel derivatives possessing increased aqueous solubility and potential for treating cancer and blood diseases.

The synthesis and biological activities of acidic, basic and neutral types of butyric acid (BA) prodrugs possessing increased aqueous solubility are described. The compounds are butyroyloxyalkyl derivatives of carboxylic acids, which possess functionalities suitable for aqueous solubilization. The anticancer activity of the prodrugs in vitro was evaluated by examining their effect on the growth of human colon, breast and pancreatic carcinoma cell lines, and their solubility in aqueous media was determined. The most promising compounds, with respect to activity and solubility, were found to be the butyroyloxymethyl esters of glutaric 2a and nicotinic acids 4a and phosphoric acid as its diethyl ester 10a, which displayed IC(50) values of 100 microM or lower. These prodrugs are expected to release formaldehyde upon metabolic hydrolysis. The corresponding butyroyloxyethyl esters (2b, 4b and 10b) that release acetaldehyde upon metabolism were significantly less potent. A similar correlation was observed for growth inhibition of the human prostate carcinoma cell lines PC-3 and LnCap and for induction of differentiation and apoptosis in the human myeloid leukemia cell line HL-60. The higher biological activity of the formaldehyde-releasing prodrugs 2a and 10a was further confirmed when induction of hemoglobin (Hb) synthesis in the human erythroleukemic cell line K562 was measured. Moreover, a therapeutic index (IC(50)/ED(50)) of ca. 5 was observed. The acute i.p. toxicity LD(50) in mice for 2a, 2b, 10a and 10b was similar and in the range of 400-600 mg kg(-1). The results obtained support the potential use of the butyric acid prodrugs for the treatment of neoplastic diseases and beta-globin disorders.

[The radiotherapeutic effect of the CM-5 fraction of turtle spleen extract]. / Radioterapevticheskii éffekt fraktsii CM-5 ékstrakta selezenki cherepakhi.

CM-5 fraction of tortoise spleen extract injected after irradiation in dose CD50/30 (540 R) raises a survival of mice to 73.4%. The number of endocolonies of spleen increases to 4.0 in experiment against 0.3 in control and the average life of experimental animals increases to 12.7 days against 8.8 in control. In vitro it has been found a considerable increase of RNA-synthetic activity of bone marrow cells stimulated by CM-5 fraction in post-irradiation period. The dose alteration factor for CM-5 fraction is 1.46 according to CD50/30 standard at survival test.

[Effect of freon-114B2 (1,2-dibromotetrafluoroethane) on the seasonal rhythm of energy activity in rat liver mitochondria]. / Vliianie freona 114B2 (1,2-dibromtetraftoretana) na sezonnyi ritm energeticheskoi aktivnosti mitokhondrii pecheni krys.

In the experiments with white rats it was found, that the inhalation of freon 114B2 at CL16, CL50, CL84 levels caused the changes in seasonal values of the energy activity in mitochondria. The action of the freon 114B2 caused disturbances in regulation of the mitochondria activity and desynchronization of seasonal rhythms of their energy activity.

[The effect of a U-2 fraction of a tortoise spleen extract on the formation of an ectopic locus of hematopoiesis]. / Vliianie fraktsii U-2 ékstrakta selezenki cherepakhi na formirovanie éktopicheskogo ochaga krovetvoreniia.

The implantation of a bone marrow fragment of intact mouse donors below the kidney capsule of irradiated (7 Gy) recipients leads to the formation of the haemopoiesis locus that somewhat exceeds, by mass and cellularity, the new-formed locus of control animals. The U-2 fraction of a tortoise spleen extract administered to recipients irradiated with the same dose increases the mass and cellularity of the haemopoiesis locus by 2.2 and 4.9 times respectively.

Scanning electron microscopic study of blood cell surfaces of the middle asian tortoise, Testudo horsfieldi.

The micromorphology and surface topography of spleen cells of the Middle Asian tortoise, Testudo horsfieldi, have been examined by scanning electron microscopy. Spleen cells comprise a population with surface characteristics similar to counterparts in mammalian spleen except for an, as yet undescribed, lymphocyte which is designated as lamellar type on the basis of its surface contours.

Regulation of trout gill AMP deaminase by lipid bilayers. Effects of phospholipid composition and temperature.

Trout gill AMP deaminase is inhibited by liposomes made of synthetic phosphatidylcholines containing higher saturated fatty acids. A preincubation of 1 hr, at 4 degrees C, was necessary to obtain the maximal effect. At 4 or 25 degrees C, these phospholipids modified essentially the substrate affinity of the enzyme by increasing the Michaelis constant proportionally to the length of the fatty acid chain. At 13 degrees C, the liposomes decreased the Hill coefficient also, thus inducing a negative cooperativity. Natural phosphatidylcholine and phosphatidylserine were without significant effect on gill AMP deaminase while natural sphingomyelin exhibited a similar effect to that shown in the presence of synthetic phosphatidylcholines. These results are discussed in relation to a possible effect of sphingomyelins in vivo.

The effect of phospholipid bilayers on AMP-deaminase from rat tissues.

The ATP-activated adenylate deaminase (EC 3.5.4.6) from the rat heart, kidney, spleen, liver and brain showed a mixed-type allosteric activation under the influence of phosphatidylcholine-containing liposomes, but no effect was observed on the non-activated enzyme isolated from all these tissues but the brain. The brain enzyme was activated by the liposomes also in the absence of ATP. No effect of the phosphatidylcholine-containing liposomes could be shown on the adenylate deaminase isolated from skeletal muscle and red blood cells. The phosphatidate-containing liposomes exerted an inhibitory effect on the enzyme isolated from the heart, kidney and brain, being without effect on the enzyme from skeletal muscle.

Purification and regulatory properties of pig kidney AMP deaminase.

AMP deaminase (EC 3.5.4.6) from pig kidney was purified about 1200-fold by chromatography on cellulose phosphate. The enzyme showed a sigmoid-shaped substrate saturation curve which was converted to hyperbolic by addition of ATP. The ATP-activated enzyme was sensitive to phosphatidylcholine-containing liposomes which caused a further increase of activity by lowering the S0.5 value and increasing V max. In the absence of ATP, the enzyme was not sensitive to phosphatidylcholine-containing liposomes. Phosphatidate-containing liposomes exerted an inhibitory effect both in the presence and absence of ATP. In the presence of ATP phosphatidate was a non-competitive inhibitor. Orthophosphate was found to be a competitive inhibitor of AMP deaminase from pig kidney. When the phosphatidylcholine/phosphatidic acid ratio in liposomes was 2.7, AMP deaminase was activated, whereas when this ratio dropped below 2.1, liposomes exerted a non-competitive inhibitory effect.

Modification by liposomes of the adenosine triphosphate-activating effect on adenylate deaminase from pig heart.

Adenylate deaminase (AMP deaminase, EC 3.5.4.6) of a high substrate specificity was purified from pig heart by chromatography on cellulose phosphate. The enzyme shows a co-operative binding of AMP [h (Hill coefficient) 2.35, with SO.5 (half-saturating substrate concentration) 5mM]. ATP and ADP act as positive effectors, lowering h to 1.55 and SO.5 to 1 mM. The addition of liposomes (phospholipid bilayers) to ATP-activated or ADP-activated enzyme causes a further shift of the h value to 1.04 and SO.5 to 0.5 mM. For ATP-activated enzyme the addition of liposomes increases Vmax. by about 100%, and for ADP-activated enzyme by 50%. Liposomes have no effect on the kinetics of AMP deaminase in the absence of ATP and ADP, and neither do they influence the inhibitory effect of orthophosphate on heart muscle AMP deaminase. Metabolic implications of these findings are discussed.