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31.
V. E. Yurinskaya T. S. Goryachaya A. A. Rubashkin A. V. Shirokova A. A. Vereninov 《Cell and Tissue Biology》2010,4(5):457-463
The K+, Na+, and Cl− balance and K+ (Rb+) and 36Cl− fluxes in U937 cells induced to apoptosis by 0.2 or 1 μM staurosporine were studied using flame emission and radioisotope
techniques. It is found that two-thirds of the total decrease in the amount of intracellular osmolytes in apoptotic cells
is accounted for by monovalent ions and one-third consists of other intracellular osmolytes. A decrease in the amount of monovalent
ions results from a decrease in the amount of K+ and Cl− and an increase in the Na+ content. The rate of 36Cl−, Rb+ (K+), and 22Na+ equilibration between cells and the medium was found to significantly exceed the rate of apoptotic change in the cellular
ion content, which indicates that unidirectional influxes and effluxes during apoptosis may be considered as being in near
balance. The drift of the ion flux balance in apoptosis caused by 0.2 μM staurosporine was found to be associated with the
increased ouabain-resistant Rb+ (K+) channel influx and insignificantly altered the ouabain-sensitive pump influx. Severe apoptosis induced by 1 μM staurosporine
is associated with reduced pump fluxes and slightly changed channel Rb+ (K+) fluxes. In apoptotic cells, the 1.4–1.8-fold decreased Cl− level is accompanied by a 1.2–1.6-fold decreased flux. 相似文献
32.
Intramembrane charge movement was recorded in guinea pig ventricular myocytes at 19-22 degrees C using the whole-cell patch clamp technique. From a holding potential of -110 mV, the dependence of intramembrane charge moved on test voltage (Q(V)) followed the sum of two Boltzmann components. One component had a transition voltage (V) of -48 mV and a total charge (Qmax) of congruent to 3 nC/microF. The other had a V of -18 mV and a Qmax of 11 nC/microF. Ba2+ currents through Ca channels began to activate at -45 mV and peaked at congruent to -15 mV. Na+ current peaked at -35 to -30 mV. Availability of charge (in pulses from -70 to +10 mV) depended on the voltage of conditioning depolarizations as two Boltzmann terms plus a constant. One term had a V of -88 mV and a Qmax of 2.5 nC/microF; the other had a V of -29 mV and a Qmax of 6.3 nC/microF. From the Q(V) dependence, the voltage dependence of the ionic currents, and the voltage dependence of the availability of charge, the low voltage term of Q(V) and availability was identified as Na gating charge, at a total of 3.5 nC/microF. The remainder, 11 nC/microF, was attributed to Ca channels. After pulses to -40 mV and above, the OFF charge movement had a slow exponentially decaying component. Its time constant had a bell-shaped dependence on OFF voltage peaking at 11 ms near -100 mV. Conditioning depolarizations above -40 mV increased the slow component exponentially with the conditioning duration (tau approximately equal to 480 ms). Its magnitude was reduced as the separation between conditioning and test pulses increased (tau approximately equal to 160 ms). The voltage distribution of the slow component of charge was measured after long (5 s) depolarizations. Its V was -100 mV, a shift of -80 mV from the value in normally polarized cells. This voltage was the same at which the time constant of the slow component peaked. Qmax and the steepness of the voltage distribution were unchanged by depolarization. This indicates that the same molecules that produce the charge movement in normally polarized cells also produce the slow component in depolarized cells. 100 microns D600 increased by 77% the slow charge movement after a 500-ms conditioning pulse. These results demonstrate two classes of charge movement associated with L-type Ca channels, with kinetics and voltage dependence similar to charge 1 and charge 2 of skeletal muscle.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
33.
D.?V.?YanvarevEmail author E.?A.?Shirokova Yu.?S.?Skoblov 《Russian Journal of Bioorganic Chemistry》2005,31(4):357-361
The kinetics of 3′-azido-3′-deoxythymidine phosphorylation with [32P]orthophosphoric acid was studied in the presence of various coupling agents. The most effective method, with the use of BrCN, provided the isolation of the target 3′-azido-3′-deoxythymidine 5′-[32P]monophosphate in 46% yield and with high specific radioactivity (>100 Ci/mmol).__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 4, 2005, pp. 399–403.Original Russian Text Copyright © 2005 by Yanvarev, Shirokova, Skoblov. 相似文献
34.
Shirokova EA Ias'ko MV Khandazhinskaia AL Ivanov AV Ianvarev DV Skoblov IuS Proniaeva TR Fediuk NV Pokrovskiĭ AG Kukhanova MK 《Bioorganicheskaia khimiia》2004,30(3):273-280
New 5'-alkyl ethoxy- and aminocarbonylphosphonates of 3'-azido-3'-deoxythymidine (AZT) were synthesized, and their antiviral properties in HIV-1-infected cell cultures and stability to chemical hydrolysis were studied. The AZT 5'-aminocarbonylphosphonates were shown to be significantly more stable in phosphate buffer (pH 7.2) than the corresponding ethoxycarbonylphosphonates. The therapeutic (selectivity) index of some of the compounds exceeded that of the parent AZT due to their higher antiviral activity. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 3; see also http://www.maik.ru. 相似文献
35.
Shirokova E Schmiedeberg K Bedner P Niessen H Willecke K Raguse JD Meyerhof W Krautwurst D 《The Journal of biological chemistry》2005,280(12):11807-11815
Olfactory receptors are the largest group of orphan G protein-coupled receptors with an infinitely small number of agonists identified out of thousands of odorants. The de-orphaning of olfactory receptor (OR) is complicated by its combinatorial odorant coding and thus requires large scale odorant and receptor screening and establishing receptor-specific odorant profiles. Here, we report on the stable reconstitution of OR-specific signaling in HeLa/Olf cells via G protein alphaolf and adenylyl cyclase type-III to the Ca2+ influx-mediating olfactory cyclic nucleotide-gated CNGA2 channel. We demonstrate the central role of Galphaolf in odorant-specific signaling out of OR. The employment of the non-typical G protein alpha15 dramatically altered the odorant specificities of 3 of 7 receptors that had been characterized previously by different groups. We further show for two OR that an odorant may be an agonist or antagonist, depending on the G protein used. HeLa/Olf cells proved suitable for high-throughput screening in fluorescence-imaging plate reader experiments, resulting in the de-orphaning of two new OR for the odorant (-)citronellal from an expression library of 93 receptors. To demonstrate the G protein dependence of its odorant response pattern, we screened the most sensitive (-)citronellal receptor Olfr43 versus 94 odorants simultaneously in the presence of Galpha15 or Galphaolf. We finally established an EC50-ranking odorant profile for Olfr43 in HeLa/Olf cells. In summary, we conclude that, in heterologous systems, odorants may function as agonists or antagonists, depending on the G protein used. HeLa/Olf cells provide an olfactory model system for functional expression and de-orphaning of OR. 相似文献
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39.
Maxim Jasko Alexander Shipitsin Elena Shirokova Alexander Krayevsky Bruce Polsky Penny Baron 《Nucleosides, nucleotides & nucleic acids》2013,32(8):879-893
Abstract 5′-Phosphonates of natural 2′-deoxynucleosides and ribonucleosides were synthesized by condensation of 3′-O-acylated 2′-deoxynucleosides or 2′,3′-substituted (2′,3′-O-isopropylidene, 2′,3′-O-methoxymethylene or 2′,3′-O-ethoxymethylene) ribonucleosides. As condensing agents, either N,N′-dicyclohexylcarbodiimide or 2,4,6-triisopropylbenzenesulphonyl chloride were used. Nucleoside 5′-ethoxycarbonylphosphonates were converted into corresponding nucleoside 5′-aminocarbonylphosphonates by action of ammonia in methanol or aqueous ammonia. 5′-Hydrogenphosphonothioates of thymidine and 3′-deoxythymidine were obtained by reaction of phosphinic acid in the presence of pivaloyl chloride with 3′-O-acetylthymidine or 3′-deoxythymidine, respectively, followed by addition of powedered sulfur. 5′-O-methylenephosphonates of thymidine and 2′-deoxyadenosine were prepared by intramolecular reaction of corresponding 3′-O-iodomethylphosphonates under basic conditions. All compounds were tested for inhibition of several viruses, including HSV-2 and CMV, but showed no activity. A few compounds insignificantly inhibited HIV-1 reproduction. Thymidine 5′-hydrogenphosphonate neutralized anti-HIV action of 3′-azido-3′-deoxythymidine (AZT) and it indirectly showed that even some nucleoside 5′-phosphonates could be partly hydrolyzed in cell culture to corresponding nucleosides. 5′-Phosphonates of modified 2′-deoxynucleosides in which one group in a phosphate residue is substituted for hydrogen, alkyl or other groups, have shown to be potent biologically 相似文献
40.
J. Matulic-Adamic I. Rosenberg A. A. Arzumanov N. B. Dyatkina E. A. Shirokova A. A. Krayevsky 《Nucleosides, nucleotides & nucleic acids》2013,32(10):1085-1092
Abstract Treatment of 3′-fluoro-3′-deoxythymidine (FLT), 3′-azido-3′-deoxythymidine (AZT) and 2′,3′-dideoxyadenosine (ddA) with tris(1,1,1,3,3,3-hexafluoro-2-propyl)phosphite or phosphorous acid and N,N'-dicyclohexylcarbodiimide produced the corresponding nucleoside 5′-hydrogenphosphonates. Reaction of FLT, AZT and 3′-deoxythymidine (ddT) with fluorophosphoric acid and 2,4,6-triisopropylbenzenesulfonyl chloride lead to the corresponding nucleoside 5′-phosphorofluoridates also on a multi-gram scale. All the compounds were isolated in high pure state by chromatographic technique. 相似文献