[Mercury ions inhibit human neutrophil apoptosis induced by ultraviolet C irradiation]. / Iony rtuti ingibiruiut apoptoz neitrofilov cheloveka, indutsirovannyi ul'trafioletovym izlucheniem diapazona C.

The action of ultraviolet C (UVC) and mercury ions (HgCl2) on human neutrophil apoptosis was investigated by flow cytometry. It is shown that HgCl2 (5-10 microM) inhibits UVC-dependent acceleration of neutrophil apoptosis but does not inhibit spontaneous apoptosis.

Steroid-induced conformational changes of FITC-labelled sarcoplasmic reticulum Ca2+-ATPase.

Interactions between transmembrane and cytoplasmic domains of Ca2+-ATPase from sarcoplasmic reticulum (SR) have been studied. To affect the hydrophobic transmembrane domain, we used four amphiphilic steroids - esters of a dibasic acid and 20-oxypregnene. All four steroids contained cholesterol-like nuclei and differed by the structure of side chains. Steroids with carboxyl groups in the side chains inhibited the rates of ATP hydrolysis and Ca2+ transport, whereas a steroid without the carboxyl group did not appreciably affect Ca2+-ATPase function. Fluorimetric titration of FITC-labelled Ca2+-ATPase in SR vesicles by Nd3+ showed that steroids increased the apparent dissociation constant for Nd3+ bound to the hydrolytic site, the potency order of the steroids being the same as for the sterol-induced inhibition of the hydrolytic activity of Ca2+-ATPase. These results suggest structural changes in the active site. Ca2+ transport was inhibited more efficiently by steroids than the hydrolytic activity of the enzyme. This could be partially due to the increase of the membrane passive permeability induced by steroids, which, in turn, reflected the efficiency of the interaction of the steroids with lipid bilayers. The effects of the steroids were largely dependent on their amphiphilicity (the availability of polar groups in regions A and D), the structure of the side chains, and, possibly, on the distance between the molecular polar groups. We suggest that the inhibition of hydrolytic and transport functions of Ca2+-ATPase in the SR membrane is due to the interaction of the steroids with the transmembrane alpha-helical segments.

[Effect of urea on conformational changes in the active site of native and reconstituted Ca-ATPase of the sarcoplasmic reticulum]. / Vliianie mocheviny na konformatsionnye perestroiki v aktivnom tsentre nativnoi i rekonstruirovannoi Ca-ATFazy sarkoplazmaticheskogo retikuluma.

The effect of urea (1-3 M) on conformational changes in the active center of native and reconstituted Ca-ATPase of sarcoplasmic reticulum modified by fluorescein-5-isothiocyanate (FITC) was studied using the method of fluorescence titration by neodymium (Nd3+) ions. Based on the analysis of curves of fluorescence quenching of FITC-labeled Ca-ATPase by Nd3+ ions, the parameters characterizing the structural changes in the Mg-ATP binding center were determined. It was assumed that FITC and Nd3+ ion bind to different polypeptide fragments moving relative to each other, which provides the conformational lability of the nucleotide binding site at some stages of the catalytic cycle. A comparison of structural changes caused by urea at the active site of native and reconstituted Ca-ATPase of sarcoplasmic reticulum indicates that the Nd3+ binding center is localized in the region of contacts of monomers in the oligomer.

[Apoptosis dynamics in human neutrophils induced by ultraviolet C irradiation during lipopolysaccharide and zinc exposure]. / Dinamika apoptoza neitrofilov cheloveka, indutsirovannogo ul'trafioletovym izlucheniem diapazona C, pri deistvii lipopolisakharida i tsinka.

The action of ultraviolet radiation with lambda = 254 nm (UVC), zinc and lipopolysaccharide on the apoptosis of human neutrophils was investigated by flow cytometry. It was shown that zinc (0.2-1 mM) inhibits the UVC-dependent acceleration of neutrophil apoptosis. Preliminary treatment with UVC cancels the inhibition of neutrophil apoptosis by lipopolysaccharide.

[Activation of poly(ADP-ribose)-polymerase inhibits apoptosis of thymocytes, caused by short-wave ultraviolet radiation]. / Aktivatsiia poli(ADF-ribozo)-polimerazy ingibiruet apoptoz timotsitov, vyzvannyi korotkovolnovym ul'trafioletovym izlucheniem.

The effect of (i) aphidicolin, a specific inhibitor of delta- and epsilon-polymerases, and nucleotide excision repair; (ii) 3-aminobenzamide, an inhibitor of poly(ADP-ribose) polymerase and base excision repair; and (iii) actinomycin D and cycloheximide, inhibitors of protein and RNA synthesis, respectively, on the induction of suppression of apoptosis of rat thymocytes by different doses of short-wavelength ultraviolet radiation was studied by flow cytometry. 3-Aminobenzamide suppressed the inhibition of apoptosis induced by the doses of short-wavelength ultraviolet radiation higher than 20 J/m2, increasing the cell death to a maximum. Thus, the inhibition of apoptosis by high short-wavelength ultraviolet radiation doses depends on the status of poly(ADP-ribose) polymerase and is prevented by 3-aminobenzamide. As opposed to 3-aminobenzamide, aphidicolin did not affect the cell death at short-wavelength radiation doses higher than 10 J/m2 but induced the apoptosis of unirradiated cells and cells irradiated with short-wavelength ultraviolet radiation doses lower than 10 J/m2. The inhibitors of protein and RNA synthesis cycloheximide and actinomycin D prevented the induction of apoptosis caused by low and medium doses but did not abolish the apoptosis-inhibiting activity of high doses of short-wavelength ultraviolet radiation.

[Modulation of human peripheral blood neutrophil apoptosis by ultraviolet C-range radiation and by gamma-irradiation]. / Moduliatsiia apoptoza neitrofilov perifericheskoi krovi cheloveka ul'trafioletovym izlucheniem diapazona C i gamma-izlucheniem.

It was found that the irradiation with in vitro UVC (254 nm) in the dose range of 6-600 J/m2 accelerates the apoptosis of human peripheral blood neutrophils in a dose-dependent manner, with saturation occurring at UVC doses of 250-300 J/m2. gamma-Irradiation with a dose of 2 Gy accelerates the apoptosis of neutrophils, whereas the irradiation with doses of 10 and 20 Gy suppresses it (by 9 h of cultivation). Lipopolysaccharide (1 microgram/ml) suppresses the UVC-induced apoptosis of neutrophils.

[Conformational changes at the ATP-catalytic site of the reconstituted sarcoplasmic reticulum Ca-ATPase under th action of pH, Ca2+, and lanthanides]. / Konformatsionnye izmeneniia v aktivnom tsentre rekonstruirovannoi Ca- ATFazy sarkoplazmaticheskogo retikuluma pod deistviem pH, Ca2+ i lantanidov.

Conformational changes at the ATP-catalytic site of the sarcoplasmic reticulum Ca-ATPase reconstituted in proteoliposomes have been studied by the fluorescence of the fluorescein 5-isothiocyanate (FITC). It binds to Lys-515 at the adenine binding site of the nucleotide domain. The FITC-Ca-ATPase fluorescence parameters have been examined in the pH range 5,7-8,0 in the presence of EGTA, Ca2+, lantanides. The quantitative method was used to calculate the equilibrium between the protein conformers E1 and E2. It is based on the analysis of fluorometric titration curves. Lantanides were used to estimate the distances between nucleotide and phosphorylation domains in the pH range 5.7-8.0. The distance between Nd(3+)-FITC was estimated to be about 1 nm at pH 6 and 1.7 nm at pH 8, which can be interpreted as an increase in the distance between the nucleotide and phosphorylation domains of Ca-ATPase in alkaline media. These studies suggest that the ligand stabilized by the E1-form of Ca(2+)-ATPase can exist in two conformational states with the closed and opened interdomain cleft in the pH range 5.7-8.0. The pH-dependence of the ratio of these states correlates with that of the E1<-->E2 equilibrium without ligands. These dependences were approximated by simple Henderson-Hasselbach equations with pK 7.0 +/- 0.1, i.e. the transition between the two protein conformations is probably governed by one proton dissociation. Model experiments were used to determine the lantanide binding with proteoliposome lipid part. The Nd3+ association constant at the substrate site has been estimated to be 1.5.10(5)M-1 at pH 6.0; 1.0.10(5) M-1 at pH 7.0 and 0.7.10(5) M-1 at pH 8.0.

[Induction and inhibition of rat thymocyte apoptosis by ultraviolet irradiation]. / Induktsiia i podavlenie apoptoza v timotsitakh krysy ul'trafioletovym oblucheniem.

Flow cytometry was used to investigate the effect of 254 nm wavelength UV irradiation on single rat thymocytes suspension in vitro. The induction of apoptosis has been observed in dose range less 20 J/m2. More high UV doses inhibited thymocyte apoptosis induced by dexamethasone, ionizing radiation and by UV itself. The necessity of protein kinase C activation has been demonstrated for realization of such dual effect of UV (apoptosis induction and suppression).

[The role of histidine residues in conformational changes in the sarcoplasmic reticulum Ca-ATPase active site]. / Rol' gistidinovykh ostatkov v konformatsionnykh izmeneniiakh v aktivnom tsentre Ca-ATFazy sarkoplazmaticheskogo retikuluma.

Conformational pH-induced changes of Mg-ATP binding site of the sarcoplasmic reticulum Ca-ATPase (SR-ATPase) were investigated by fluorescence energy transfer between covalently bound fluorescent label (fluorescein-5-isothiocyanate, FITC) and lanthanide ion (Nd3+). These changes were approximated by simple Henderson-Hasselbach equation with the apparent pK 7.0 +/- 0.1 which is similar that of a histidyl residue [3]. In this work it was used the double chemical modification of SR-ATPase to research the role of histidyl residues in this conformational transition. Diethyl pyrocarbonate was used to modify the histidyl residues of the SR-ATPase. The influence of histidyl modification on the functional parameters (the rates of ATP and p-nitrophenyl phosphate hydrolysis, the Ca transport and the level of Ca2+ accumulation) was monitored by the fluorescent probes (Quin-2, chlortetracycline) using fluorescent, spectrophotometric and pH-metric measurements. In the result of these experiments it was found the appropriate conditions to carry out the second modification. The DEPC-SR-ATPase was labeled by FITC. The pH-dependent conformational changes in the active site of FITC-DEPC-SR-ATPase were studied by the method of the fluorescence energy transfer between FITC and Nd3+ in the region of pH 6-8. The histidyl modification of FITC-DEPC-SR-ATPase resulted in the significant shift of the curve of fluorescence energy transfer efficiency (the apparent pK > 7.5). These results suggest that the conformational transition in the active site of SR-ATPase was controlled by the histidyl residues.

The use of flow cytometry for the investigation of cell death.

Flow cytometry is more and more widely used for investigations of cell death, predominantly in the study of DNA degradation in cells dying by apoptosis. There are different interpretations of changes observed in DNA histograms of these cells. We describe an approach based on extraction of chromatin degradation products from fixed cells and subsequent staining with DNA specific dyes. Apoptotic cells containing fragmented DNA are observed in < 2C DNA region of DNA histograms. DNA histograms of irradiated thymocytes dying in vitro and stained without extraction of fragmented DNA do not differ from control. Under the same staining conditions DNA histograms of lymphocytes dying in thymus of irradiated animals reveal fluorescent material in < 2C DNA region, most likely due to formation of apoptotic bodies (cell fragments, some of them contain fragments of nuclei). Similar changes are observed in thymocytes dying upon glucocorticoid treatment. Our present results and other data indicate that reduced amount of DNA in dying cells is the main reason for changes of DNA histograms. Examples of application of the method described for the investigations of cell death modifiers are presented.

Effects of pH, Ca2+ and lanthanides on conformation of the sarcoplasmic reticulum Ca(2+)-ATPase catalytic site.

The conformational changes at the ATP-catalytic site of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase have been studied by the fluorescence of the fluorescein 5-isothiocyanate (FITC) bound to the adenine subsite. The FITC-SR fluorescence parameters have been examined in the pH range 5.7-8.0 in the presence of EGTA, Ca2+ or Ln3+ (La3+, Pr3+, Nd3+, Tb3+, etc.). A quantitative method to calculate the equilibrium between the protein conformers is proposed on the basis of the fluorometric titration curve analysis. The distance Nd(3+)-FITC was estimated to be about 1 nm at pH 6-7 and 1.7 nm at pH 8 which can be interpreted as an increase of the distance between the nucleotide and phosphorylation domains of Ca(2+)-ATPase in alkaline media. These studies suggest that the ligand-stabilized E1-form of Ca(2+)-ATPase can exist in two conformational states with the closed and opened interdomain cleft in the pH range 5.7-8.0. The pH-dependence of the ratio of these states correlates with that of the E1----E2 equilibrium without ligands. These dependences were approximated by simple Henderson-Hasselbach equations with pK 7.0 +/- 0.1, i.e. the transition between two protein conformations is probably governed by one proton dissociation.

[Effective reconstitution of sarcoplasmic reticulum Ca2+-ATPase using lubrol PX]. / Effektivnaia rekonstruktsiia Ca2+-ATP-azy sarkoplazmaticheskogo retikuluma s ispol'zovaniem lubrola PX.

Ca2(+)-ATPase of sarcoplasmic reticulum was reconstituted in the proteoliposomes by the salting out procedure. Triton X-100, C12E8 and Lubrol PX were used for the solubilization of the Ca2(+)-ATPase. Using fluorescent probes (diS-C3-(5), chlortetracycline) as well pH-measuring method, the functional of the reconstituted Ca2(+)-ATPase was comparatively studied in three types of proteoliposomes. The efficiency of Ca2(+)-ATPase grew in the following detergent order: Triton X-100, C12E8, Lubrol PX.

[The effect of electromagnetic radiation on the membranes of the sarcoplasmic reticulum]. / Vliianie élektromagnitnogo izlucheniia na membrany sarkoplazmaticheskogo retikuluma.

On the sarcoplasmic reticulum membranes has been shown that at temperature of Ca2(+)-ATPase activity change of dependence in the Arrhenius plot the microwaves (2450 MHz, specific absorption rate 12 w/kg) inhibit the ATP-hydrolase and Ca2(+)-transporting activity of Ca2(+)-ATPase. The effect of radiation exhibits within the narrow temperature range (approximately 1 degree C) and quantitatively corresponds to the decrease of Ca2(+)-ATPase activity caused by the decrease of temperature by 1.6 degrees C from 18 degrees C. The fluorescence intensity of naphthalene sulfonic probes reduces under the influence of microwaves at 18 degrees C.

[General similarities and differences in the postradiation death of various animal species and of man]. / Obshchie zakonomernosti i razlichiia postradiatsionnoi gibeli limfotsitov raznykh vidov zhivotnykh i cheloveka.

It was found in various animal species and man that an ordered internucleosome fragmentation of DNA is characteristic of lymphoid cells dying in the interphase. Both in vivo and in vitro, the postirradiation DNA degradation in thymocytes of rodents and piglets preceded the increase in the permeability of their plasma membrane. The in vivo kinetics of death of lymphoid cells from the thymus and spleen is similar in rodents and piglets. Rat thymocytes died in vitro earlier than thymocytes of piglets, calves and man which was evidently associated with a worse adaptive capacity of the latter to cultivation conditions.

[The patterns of colchicine-induced death of lymphoid cells]. / Zakonomernosti indutsirovannoi kolkhitsinom gibeli limfoidnykh kletok.

It is shown that colchicine injection at doses higher than 1 mg/kg of animal weight induces cell death in thymus, spleen, bone marrow and intestine mucosa. The cell death is accompanied by a regular internucleosomal cleavage of nuclear DNA and by the elimination of the formed fragments from cells. Both the processes begin after a 1.5 hour lag-period and proceed before the outer membrane permeability for supravital dyes increases. DNA degradation is prevented by the inhibitor of protein synthesis cycloheximide. Cytochalasin B does not induce chromatin degradation or cell death and has no effect on radiation death of lymphocytes. A possible role of microtubule destruction as a switch-on mechanism of DNA degradation and cell death is discussed.

[Sarcoplasmic reticulum Ca2+-ATPase reconstructed into low-permeable proteoliposomes]. / Issledovanie Ca2+-ATP-azy sarkoplazmaticheskogo retikuluma, rekonstruirovannoi v nizkopronitsaemye proteoliposomy.

The kinetic characteristics of Ca2+-ATPase reconstructed into proteoliposomes were studied with fluorescent probes. Reconstruction was made using purified resin XAD-2. The data obtained evidence for an electrogenic character of the reconstructed Ca2+-ATPase activity.

Interaction of the voltage-sensing fluorescent probe diS-C3-(5) with dipalmitoylphosphatidylcholine liposomes.

The interaction of the probe diS-C3-(5) with dipalmitoylphosphatidylcholine (DPPC) liposomes has been studied using fluorescence and differential scanning calorimetry (DSC). The partition coefficients (K) of the probe for the lipid and the aqueous phase (in terms of molar part units) were (1.20 +/- 0.4) X 10(6) at 45 degrees C and (0.50 +/- 0.07) X 10(6) at 23 and 36 degrees C. In terms of volume concentration units, these values correspond to Kp = (2.88 +/- 0.10) X 10(4) and Kp = (1.20 +/- 0.17) X 10(4), respectively. DSC thermograms were practically identical both for large unilamellar and multilamellar liposomes. The main transition peak remained practically unchanged over the entire range of the probe concentrations used. The pretransition could be observed up to maximal probe concentrations applied and it widened and shifted from 35.4 degrees C in pure DPPC to approximately 32 degrees C at a probe/lipid ratio of 0.027. These results suggest that in both quasicrystalline and liquid crystalline lipid bilayers the probe molecules are included in "defects" between structurally ordered microregions (microdomains or clusters). The dependence of the fluorescence response on the transmembrane potential in a suspension of unilamellar DPPC vesicles suggest that the equilibrium thermodynamic model is valid for liquid crystalline bilayers.

[Determination of the radiosensitivity of interphase-dying cells in the thymus, spleen and bone marrow of rats by flow cytometry]. / Opredelenie radiochuvstvitel'nosti interfazno gibnushchikh kletok timusa, selezenki i kostnogo mozga krys metodom protochnoi tsitometrii.

The flow cytofluorometry of cells stained with a DNA-specific probe was used to determine the share of dying cells (containing less than 2C DNA) in thymus, spleen and bone marrow cells of irradiated rats. The cell death curves for spleen and bone marrow had a plateau by the 6th h, and for thymus, by the 10th h following irradiation with different doses. On the basis of the dose-response relationship the share of cells dying in the interphase was determined in each organ under study, and dose-response curves shaped. All the curves had no shoulder. Do was 3.0, 3.0 and 3.7 Gy for thymus, spleen, and bone marrow cells, respectively.

[Quantitative analysis of the effect of cysteamine on interphase cell death by flow cytometry]. / Kolichestvennyi analiz vliianiia tsisteamina na interfaznuiu gibel' kletok metodom protochnoi tsitometrii.

The method of flow cytofluorometry was used to study the radioprotective effect of cysteamine on cells dying in the interphase. DMF was 1.67, 1.67, and 1.76 for thymus, spleen, and bone marrow, respectively.