Partial dependence of human peripheral blood leukocyte binding to high molecular weight fucoidan on divalent cations.

L-selectin (CD62L) is the principal leukocyte adhesion molecule for the high endothelial venules of peripheral lymph nodes. This adhesion has an absolute requirement for calcium ions. Nevertheless, some studies have shown carbohydrate adhesion receptor interactions on lymphocytes and neutrophils, including the L-selectin molecule, that are Ca-independent. In the present study fucoidan, a reportedly Ca2+ independent ligand of L-selectin, and Mabs to human CD62L were coupled to magnetic polystyrene beads (MPB), as a model of leukocyte-surface interactions, and the efficiency of human leukocyte separation was investigated. 30% of Ficoll-purified human mononuclear cells and 75% of dextran-purified human leukocytes (DPHL) were specifically bound by fucoidan-modified MPB in the presence of Ca2+; 55% of dextran-purified leukocytes were specifically bound in the absence of Ca2+. The specific binding was inhibited by an excess of free fucoidan. The data obtained show the presence of Ca-independent adhesion determinants, specific to fucoidan on human leukocytes. No significant specific binding of leukocytes to fucoidan-modified MPB was found after the incubation with fresh human Ca(2+)-depleted whole blood. More than 90% of DPHL were specifically bound to MPB modified with Mabs to human CD62L irrespective of Ca2+ presence. The same degree of separation was achieved after the incubation with fresh human Ca(2+)-depleted-whole blood with anti-CD62L modified beads.

[Immunomodulating effect of ecdysterones]. / Immunomoduliatornyi éffekt ékdisteronov.

Ecdysterone, its 20-desoxy-derivative alpha-ecdysone, their 2-desoxy-derivatives ecdysterone 2, 3, 22-triacetate and preparation BTI-4 have been studied for their effect on [3H]-thymidine incorporation in different populations of animal and human lymphocytes. It is shown the ecdysteron and its analogs in concentrations of 10(-12)-10(-5) M take considerable stimulating effect on DNA biosynthesis in animal lymphocytes activated by polyclonal mitogens. The concentration of ecdysterone being increased to 10(-4) m one can observe complete inhibition of activating effect of polyclonal mitogens. Effect of the studied ecdysteroids did not considerably depend on their structure. In case of splenocytes the stimulating effect of ecdysterone on DNA biosynthesis is less expressed than in the case of activated thymocytes. Ecdysterone was established to have considerable inhibiting effect on DNA biosynthesis in the culture of activated Con A cells of lymphocytes in the peripheral blood of healthy donors.

[Kinetics of the interaction of methylene diphosphonic acid and inorganic pyrophosphate with DNA-dependent RNA-polymerase from calf thymus]. / Kinetika vzaimodeistviia metilendifosfonovoi kisloty i neorganicheskogo pirofosfata s DNK-zavisimoi RNK-polimerazoi II timusa telenka.

The kinetics of interaction of PPi and its diphosphonic analog, methylenediphosphonic acid (MDPA), with nucleoside triphosphates, DNA and Mg2+ binding sites of DNA-dependent RNA polymerase II from calf thymus was investigated. The values of apparent Km in the NTP polymerization reaction for ATP and CTP equal to 2.7 X 10(-4) and 1.8 X 10(-4) M, respectively, were determined. It was shown that MDPA and PPi competitively inhibited the RNA polymerase reaction with respect to nucleoside triphosphate. The inhibition constants (Ki) of ATP and CTP incorporation for MDPA were 2.2 X 10(-4) and 3.3 X 10(-4) M, respectively, while those of the nucleoside triphosphate incorporation for PPi were equal to 1.4 X 10(-4) and 2.0 X 10(-4) M, respectively. MDPA and PPi were incompetitive inhibitors of template (DNA) and Mn2+. A possible mechanism of inhibition of the RNA polymerase reaction by MDPA is proposed.

[The influence of diphosphonic analogues of inorganic pyrophosphate on activity of RNA-polymerases from the calf thymus]. / Vliianie difosfonovykh analogov neorganicheskogo pirofosfata na aktivnost' rnk-polimeraz iz timusa telenka.

Diphosphonic analogues of inorganic pyrophosphate (PPi): methylene-, oxyethylidene-, aminomethylenediphosphonic acids as well as phosphonacetic, imidodiphosphoric bis- (phosphonomethyl)-phosphonic acids and methylenediphosphonic and phosphonic acid monoanhydrides were studied for their effect on the RNA-synthesizing activity of thymocytes. DNA-dependent RNA-polymerases I and II from the calf thymus nuclei were used for these studies. The analogues and PPi under study are shown to be inhibitors of both RNA-polymerases in nuclei from calf thymus and of purified RNA-polymerase II, which is more sensitive to the effect of diphosphonates. Methylenediphosphonic acid is the strongest inhibitor among the studied analogues, and imidodiphosphoric and phosphonacetic acids are the weakest inhibitors. Inhibition of purified RNA-polymerase II by diphosphonates has a complex character and includes both interaction of the PPi analogues with enzymes and chelating by them of Mn ions which are cofactors for RNA polymerase.

[The influence of diphosphonic analogues of inorganic pyrophosphate on reactions catalyzed by DNA-dependent RNA-polymerase II]. / Vliianie difosfonovykh analogov neorganicheskogo pirofosfata na reaktsii, kataliziruemye dnk-zavisimoi rnk-polimerazoi II.

Diphosphonic analogues of inorganic pyrophosphate were studied for their influence both on RNA pyrophosphorolysis and pyrophosphate exchange, catalyzed by purified DNA-dependent RNA-polymerase II from calf thymus. Methylene-, oxyethylene-and aminomethylenediphosphonic acids are shown to compete with PPi for incorporation into nucleoside triphosphate. They activate RNA pyrophosphorolysis in the concentration of 2 mM, but to a less extent than PPi does.