[Activation of bovine retinal rod outer segment cGMP-specific phosphodiesterase by the transducin-GTP complex in a physiologically significant range of free calcium ion concentrations].

The kinetic behavior of cGMP-specific phosphodiesterase in a totally bleached bovine retinal rod outer segment suspension was studied by the pH-metric method at high and low concentrations of free calcium ions (≈ 100 µM and 10 nM, respectively). The phosphodiesterase was activated by low GTP concentrations (about 1-2 µM) that were comparable with the concentration of G-protein transducin, its GTP-binding alpha-subunit was the intrinsic activator of photoreceptor phosphodiesterase. The results allow the suggestion that besides the earlier described system of RGS proteins, participating in the acceleration of GTP hydrolysis, rod outer segments also contain an additional Ca(2+)-dependent mechanism to inactivate so called "free transducin", i.e. active transducin that has not managed to interact with phosphodiesterase during the time, restricted by duration of photoreceptor response.

[The phosphorylation state of transducin beta-subunits].

The supposition that nucleoside diphosphate kinase is the enzyme that phosphorylates transducin beta-subunits on one of the histidine residues (His-266) has been analyzed. It stands the reason that 1) this enzyme is multifunctional and plays in particular the role of protein histidine kinase; and 2) the phosphorylated beta-subunit of transducin may activate transducin via the mechanism of transphosphorylation. Nevertheless, in our experiments, in which different forms of transducin preparations were incubated with α- and ß-isoforms of recombinant rat NDP kinase in the presence of [γ32P]ATP or [γ32P]GTP (specific activity of about 1 Ci/mmol) followed by separation of proteins by electrophoresis and-gel radio-autography, the phosphorylation of the transducin beta-subunit wasn't succeeded to be found. The negative result of our experiments most likely implies that the major part of transducin beta-subunits in the preparations has already been phosphorylated via a process that takes place in vivo.

[Involvement of cyclic adenosine monophosphate in the control of motile behavior of Physarum polycephalum plasmodium].

Possible involvement of autocrine factors into the control of motile behavior via a receptor-mediated mechanism was investigated in Physarum polycephalum plasmodium, a multinuclear amoeboid cell with the auto-oscillatory mode of motility. Cyclic adenosine monophosphate (cAMP) and extracellular cAMP-specific phosphodiesterase, its involvement into the control of plasmodium motile behavior was proved by action of its strong inhibitor, were regarded as putative autocrine factors. It was shown that the plasmodium secreted cAMP. When it was introduced into agar support, 0,1-1 mM cAMP induced a delay of the plasmodium spreading and its transition to migration. When locally applied, cAMP at the same concentrations induced typical for attractant action the increase in oscillation frequency and the decrease of ectoplasm elasticity. The ability to exhibit positive chemotaxis in cAMP gradient and the dependence of its realization were shown to depend on the plasmodium state. Chemotaxis test specimens obtained from the migrating plasmodium, unlike those obtained from growing culture, generate alternative fronts which compete effectively with fronts oriented towards the attractant increment. The results obtained support our supposition stated earlier that advance of the Physarum polycephalum plasmodium leading edge is determined by local extracellular cAMP gradients arising from a time delay between secretion and hydrolysis of the nucleotide.

[Purification of transducin betagamma-subunit complex from isotonic extracts of bovine retinal rod outer segments].

A method for obtaining a free complex of transducin betagamma-subunits from bovine retinal rod outer segments in a highly purified state has been suggested.

[Involvement of extracellular cAMP-specific phosphodiesterase in control of motile activity of Physarum polycephalum plasmodium].

Possible involvement of extracellular cAMP-specific phosphodiesterase in the control of cell motile behavior has been investigated in Physarum polycephalum plasmodium, a multinuclear amoeboid cell with the autooscillatory mode of motility. It was found that the rate of the hydrolysis of 10 mM cAMP by a partially purified preparation of cAMP-specific phosphodiesterase secreted by the plasmodium in the course of migration decreases 20-30 times under the action of 1 mM dithiothreitol. In the presence of 1-5 mM of this strong reducing agent, the onset of the plasmodium spreading and the transition to the stage of migration were delayed in a concentration-dependent manner. In accordance with the morphological pattern of motile behavior, the duration of the maintenance of high frequency autooscillations, which normally precede the increase in the rate of the spreading and appear also in response to the application of attractants at spatially uniform concentrations, strongly increased by the action of dithiothreitol. The results obtained suggest that the autocrine production of cAMP and extracellular cAMP-specific phosphodiesterase is an important constituent of the mechanism controlling the motile behavior of the Physarum polycephalum plasmodium.

[Viferon efficacy in influenza in adult patients].

The therapeutic efficacy of Viferon (suppositories of human recombinant interferon alfa-2) was investigated in a double-blind controlled study with the use of Arbidol as a reference drug in the treatment of patients with influenza. Viferon and Arbidol lowered the signs of the fever, intoxication, catarrh and the disease as the whole.

[Evaluation of the efficacy of wiferon and arbidol in adult influenza].

The therapeutic efficacy of wiferon (recombinant alpha2beta-interferon) versus arbidol was studied in a double-blind controlled study in patients with laboratorily verified influenza. Within the first 24-36 hours after the onset of the disease, wiferon and arbidol reduced the duration of fever, intoxication, and the catarrhal symptoms of the disease as a whole. The agents were shown to have an immunomodulating effect.

[Viferon suppositories in the treatment of influenza in adults].

One hundred and one patients at the age of 18 to 60 years suffering from influenza were observed during increased ratio of the sickness due to the influenza virus types A (H1N1 and H3N2) and B. The diagnosis of influenza was confirmed by the laboratory tests. Viferon was used in the treatment of 35 patients. The randomized double blind placebo-controlled study revealed high therapeutic efficacy ofviferon and its immunomodulating effect on the T-cells, the neutrophil phagocytic activity and the decrease of the levels of the circulating immune complexes. Viferon and arbidol decreased the fever periods and the toxicosis symptoms vs. the placebo. The therapeutic efficacies of viferon and arbidol were on the whole comparable, whereas the clinical findings and the results of the immunological tests were evident of the viferon higher therapeutic and immunomodulating efficacy. No side effects of the drugs were recorded. The tolerability was excellent. Viferon can be recommended for the treatment of influenza in adults.

[Thermostable extracellular cyclic nucleotide phosphodiesterase from Physarum polycephalum plasmodium].

The cyclic nucleotide phosphodiesterase secreted by Physarum polycephalum plasmodium into extracellular medium has been partially purified by DEAE cellulose chromatography, ultrafiltration, and HPLC. The results obtained by gel filtration, HPLC, electrophoresis, and isoelectric focusing suggest that, the native enzyme in solution is a monomer with a molecular mass of about 90 kDa and pI in the range 3.6 - 4.0. The Km values were estimated to be about 0.9 mM and 7.7 mM, respectively, and Vm for both substrates were similar (up to several thousand micromoles of cAMP hydrolyzed/hour per mg of enzyme). The partially purified enzyme was shown to be extremely stable. It did not lose the activity after heat treatment at 100 degrees C during 30 min. The enzyme was active in the presence of 1% SDS, but it was fully inactivated under the same conditions in the presence of beta-mercaptoethanol. The properties of the phosphodiesterase from Physarum polycephalum are discussed.

[Calcium-dependent interaction of transducin with calmodulin-sepharose].

It has been shown by affinity chromatography on calmodulin-sepharose that transducin, a G protein of bovine retinal rod outer segments interacts with Ca(2+)-calmodulin. This result assumes that the main part of calmodulin in dark retinal rod outer segments is associated with transducin. It has been suggested that photoactivation of retinal rods induces changes in intracellular calmodulin concentration, which may be one of the steps involved in the light adaptation of photoreceptor.

Properties and content of cyclic nucleotide phosphodiesterase in photoreceptor outer segments of ground squirrel retina.

Cyclic GMP-specific phosphodiesterase (3',5'-cyclic-nucleotide 5'-nucleotidohydrolase, EC 1.3.4.17) (PDE) is thought to be a key enzyme of the retinal-rod phototransduction cyclic nucleotide pathway. We attempted to investigate the properties and content of PDE in retinal-cone photoreceptors. The fractions obtained from cone-dominant ground squirrel retinas were analyzed for cone visual pigment content and PDE activity. The cone visual pigment content was estimated to be approx. 65 pmol per retina. The distribution of cone visual pigment coincided with that of the PDE activity through several steps of photoreceptor membrane purification by sucrose density gradient centrifugation. The ground squirrel retinal PDE was similar to the retinal-rod PDE by its kinetic properties, thermostability, sensitivity to tryptic activation, Stokes radius and pI values. The cone visual pigment enriched fractions contained the heat-stable trypsin-inactivated PDE inhibitor. Its functional properties seem to be similar to those of the retinal-rod PDE inhibitory subunit. The PDE content in ground squirrel retina was roughly estimated to be about five copies of enzyme per 100 cone visual pigment molecules. The obtained results indicated that the major portion of ground squirrel retinal cyclic GMP-specific PDE is the endogenous cone photoreceptor membrane enzyme and strongly supported the conception about the key role of PDE in cone phototransduction. The existence of essential differences between rod and cone systems rapidly returning cyclic GMP-specific amplification cascade components to the dark (or inactivated) states after photon absorption was suggested. If this suggestion is true, the well-known distinctions between response kinetics and light sensitivity of these two kinds of photoreceptor can be explained.

Reinterpretation of luminiscence properties of neurotoxins from the venom of Middle-Asian corba Naja oxiana eichw.

A new interpretation of previous work (Bukolova-Orlova, T. G., Burstein, E.A. and Yukelson, L. Ya (1974) Biochim. Biophys. Acta 342, 272-280) and some new data on the luminescence of neurotoxins I and II from Naja oxiana venom is given, based on the newer data on their complete amino acid sequences. Very effective excitation energy exchange exists between Trp-27 and Trp-33 in neurotoxin I and between Trp-27 and Trp-28 in neurotoxin II, Which results in the tryptophanyl fluorescence spectra of each of the proteins seeming to be monocomponent ones. The lowered fluorescence quantium yield value, the shortened phosphorescence lifetime (80% of the emission has tau p less than 0.5 s, 20% has tau p = 4.8 s, comparing with usual tau p = 5.5-5.9 s) and decreased phosphorescence to fluorescence ratio (0.042, as compared to the usual 0.4-0.7) for neurotoxin I suggest that the indole chromophore of Trp-27 and/or Trp-33 are in contact with heavy sulfur atoms of disulfide, most probably of Cys(28)-Cys(32). Tryptophanyls in neurotoxin II are exposed to the solvent, however their accessibility in relation to that of the free tryptophan to the negatively charged quencher I- (0.455) is much higher than that for the positively charged Cs+ (0.08), which is probably due to the proximity of cationic Lys-25, Lys-26 and His-31. The difference of accessibility to the negative and positive quenchers is even more pronounced in the case of the neurotoxin I (1.04 and 0 +/- 0.02, respectively), though in its chromophore vicinity along the primary structure there is only one cationic group, Lys-25. This fact together with the analysis of the amino acid sequence, suggest that the space folding of this polypeptide results in the close proximity of Trp-27 and/or Trp-33 with the C-terminal peptide segment 67-73, which contains four cationic groups (His-67, Lys-69, Lys-71 and Arg-72).

Transducin-mediated, isoform-specific interaction of recombinant rat nucleoside diphosphate kinases with bleached bovine retinal rod outer segment membranes.

The properties of the binding of recombinant rat nucleoside diphosphate (NDP) kinase isoforms alpha and beta (NDP kinase alpha and beta, respectively) to bleached bovine retinal rod outer segment (ROS) membranes were investigated. It was found that: (1) both NDP kinase isoforms interacted with ROS membranes in a pH-, cation- and GTPgammaS-dependent manner; (2) the retinal G-protein transducin was an obligatory factor for the interaction; (3) the apparent affinity of NDP kinase alpha for ROS membranes was about 100-fold higher than that of NDP kinase beta; and (4) an alpha-isoform-specific peptide, corresponding to the sequence of the N-terminal third (variable region), had the ability to displace bovine NDP kinase from ROS membranes. The results suggest the possible involvement of NDP kinases in cellular regulation via interaction with G-proteins and provide a structural basis for the possible differential roles of mammalian NDP kinase isoforms in the cell.

Overexpression of nucleoside diphosphate kinases induces neurite outgrowth and their substitution to inactive forms leads to suppression of nerve growth factor- and dibutyryl cyclic AMP-induced effects in PC12D cells.

Whether nucleoside diphosphate kinase (NDPK) is involved in neuronal differentiation was investigated with special reference to its enzyme activity. Neurite outgrowth of PC12D cells induced by nerve growth factor or a cyclic AMP analog was suppressed to some extent when inactive NDPKs (the active site histidine 118 was replaced with alanine), not active forms, were transiently overexpressed. This suppression was more definite in their stably expressed clones. NDPKbeta-transfected clones and, to a lesser extent, NDPKalpha-transfected clones, but not inactive NDPK-transfected clones, extended neurites without differentiation inducers. These results imply that NDPKs may play a role by exerting their enzyme activity during differentiation of PC12 cells.

Comparative study of recombinant rat nucleoside diphosphate kinases alpha and beta by intrinsic protein fluorescence.

Nucleoside diphosphate (NDP) kinases of mammals are hexamers of two sorts of randomly associated highly homologous subunits of 152 residues each and, therefore exist in cell as NDP kinase isoforms. The catalytic properties and three-dimensional structures of the isoforms are very similar. The physiological meaning of the existence of the isoforms in cells remained unclear, but studying recombinant rat NDP kinases alpha and beta, each containing only one sort of subunits, we discovered that, in contrast to the isoenzyme beta, NDP kinase alpha is able to interact with the complex between bleached rhodopsin and G-protein transducin in retinal rod membranes at lowered pH values (Orlov et al. FEBS Lett. 389, 186-190, 1996). In order to search for possible molecular basis of such differences between these isoenzymes, a detailed comparative study of their intrinsic fluorescence properties in a large range of solvent conditions was performed in this work. The isoenzymes alpha and beta both contain the same three tryptophan (Trp78, 133, Ind 149) and four tyrosine (Tyr 52, 67, 147, and 151) residues per subunit, but exhibit pronounced differences in their fluorescence properties (both in spectral positions and shape and quantum yield values) and behave differently under pH titration. Whereas NDP kinase alpha undergoes spectral changes in the pH range 5-7 with the mid-point at 6.2, no unequivocal indication of a structural change of NDP kinase beta under pH titration from 9 to 5 was obtained. Since the pH dependencies obtained for fluorescence of isoenzyme alpha resembles the dependence of its binding to the rhodopsin-transducin complex it was suggested that the differences between the NDP kinase isoenzymes alpha and beta in the pH-induced behavior, revealed by the fluorescence spectroscopy, and the differences in their ability to interact with rhodopsin-transducin complex may have the same physical nature, that would be a physico-chemical reason of possible functional dissimilarity of NDP kinase isoforms in cell. An additional analysis of three-dimensional structure of homologous NDP kinases revealed that the source of the differences in fluorescence properties and pH-titration behavior between the isoenzymes alpha and beta may be due to the difference in their global electrostatic charges, rather than to any structural differences between them at neutral pH. The unusually high positive electrostatic potential at he deeply buried active site Tyr52 makes possible that it exists in deprotonated tyrosinate form at neutral and moderately acidic solution. Such a possibility may account for rather unusual fluorescence properties of NDP kinase alpha: (i) rather long-wavelength emission of NDP kinase alpha at ca. 340 nm at pH ca. 8 at extremely low accessibility to external quenchers and, possibly, (ii) an unusually high quantum yield value (ca. 0.42).

[Effect of splenic cells sensitized with staphylococcal enterotoxin A on the metastasis of Lewis lung carcinoma in mice]. / Vliianie kletok selezenki, sensibilizirovannykh stafilokokkovym énterotoksinom A, na metastazirovanie kartsinomy legkikh L'iuis u myshei.

The effect of intact mouse spleen cells sensitized in vitro with staphylococcus aureus enterotoxin A (SEA) on spreading of mouse Lewis carcinoma was studied. A significant decrease in a number of metastases in the lungs and in the weight of the lungs was observed after multiple intrapulmonary inoculation of spleen cells treated with SEA for 6 hours. The effect was less marked after inoculation of the sensitized cells intraperitoneally or into the femoral muscle of the leg affected with the tumour. After multiple inoculations of the sensitized cells, the spleen cells of the treated animals develop the state of interferon hyporeactivity to SEA but not to PHA or NDV.

[Effect of staphylococcal enterotoxin A on the development of Lewis lung carcinoma in mice]. / Vliianie stafilokokkovogo énterotoksina A na razvitie kartsinomy legkogo L'iuis u myshei.

The effect of Staphylococcus aureus enterotoxin A (SEA) was studied for its effect on the development of the Lewis carcinoma in mice. It was shown that administration of SEA immediately after the appearance of the primary node in mice after transplantation of tumour cells led to insignificant inhibition of the node growth and a slight decrease of tumour metastasizing into the lungs. Inoculation of mice after the appearance of the primary node with 1/microgram of SEA 5 times a week significantly increased their survival rate. The lack of the marked effect of SEA appears to be associated with the disturbance of the immune interferon system functioning in tumour-bearing mice, since the production of serum interferon induced by SEA in mice with tumours was considerably lower than in the intact ones.

[Isolation of mononuclear cells from the bone marrow of patients with hemoblastoses using one-step ficoll-verographin density gradient separation]. / Vydelenie mononuklearnykh kletok iz kostnogo mozga bol'nykh gemoblastozami putem razdelenia v odnostupenchatom gradiente plotnosti fikoll-verografin.

The one-step ficoll-verographin density-gradient centrifugation was first used for isolating mononuclear cells from human bone marrow. Children suffering from various hemoblastoses were donors. The morphological control of the cells isolated from the interface layer and May-Grünwald stained showed a high efficiency of the procedure proposed. Due to the nosological form of malignancy mononuclears composed from 100% (in the relapse of acute lymphocytic leukemia) to 81% (in hemoblastosis with "intact" bone marrow: lymphogranulomatosis and lymphosarcoma without leukemization) among the isolated cells. Thus, the one-step ficoll-verographin density-gradient centrifugation may be used for extensive clinical research, permitting more time-consuming procedures to be avoided.

Neuraminidase activity of influenza virus-infected cells: localization and properties.

Infection of chick embryo cell (CEC) cultures with influenza virus results in the appearance of neuraminidase activity lost by the cells as a result of cultivation. Neuraminidase activity is associated mainly with the lysomal cell fraction. Different distribution of neuraminidase activity in lysosomes with various densities, different reaction to sodium ethylene diamine tetracetate (EDTA) and differenct optimal pH suggest that at early stages of viral infection the cell enzyme is activated and by the 7th hour of infection viral neuraminidase is synthesized.

Production of alpha-interferon by human leukocytes.

We examined the capacity of human leukocytes to produce alpha-interferon subtypes following viral induction. It was shown that the beta-propiolactone inactivated viral inductor stimulated the production of a complete set of native alpha-interferon subtypes. Acid treatment of native interferon at pH 2.0 inactivated the acid-labile portion of alpha-interferon. Its exposure to 37 degrees C for 5 days and repeated acid treatment at pH 2.0 resulted in additional inactivation of some interferon pool fractions. The acid-labile subtypes of human alpha-interferon were formed in a nonadherent mononuclear cell fraction.