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11.
P F Agris R Parks L Bowman R H Guenther S A Kovacs S Pelsue 《Experimental cell research》1990,189(2):276-279
Type I topoisomerases (EC 5.99.1.2) are those enzymes capable of relaxing negatively supercoiled DNA without the need for ATP. The central role played by these enzymes in cell function suggests that the structure of type I topoisomerases may be highly conserved in eukaryotic cells. However, the extent of the conservation among eukaryotes is unknown. Human DNA topoisomerase I is an autoimmune antigen (Scl-70) of scleroderma patients. We have found that the autoimmune antibodies in human Scl-70 sera recognize protein from various plants, and these proteins display DNA relaxation function. In addition, Scl-70 antibodies were able to inhibit enzymatic activity of plant topoisomerase I. Therefore, the immunological cross-reactivity of the plant topoisomerase with human antibodies demonstrates that, despite divergence of eukaryotic organisms, these plant and animal enzymes retain structurally similar enzymatic features. 相似文献
12.
A series of biochemical investigations were undertaken to determine the mechanism of Q-banding. The results were as follows: 1. In agreement with previous studies, highly AT-rich DNA, such as poly(dA)-poly(dT), markedly enhanced quinacrine fluorescence while GC containing DNA quenched fluorescence. These effects persisted at DNA concentrations comparable to those in the metaphase chromosome. 2. Studies of quinacrine-DNA complexes in regard to the hypochromism of quinacrine, DNA Tm, DNA viscosity, and equilibrium dialysis, indicated the quinacrine was bound by intercalation with relatively little side binding. 3. Single or double stranded nucleotide polymers, in the form of complete or partial helices, were 1000-fold more effective in quenching than solutions of single nucleotides, suggesting that base stacking is required for quenching. 4. Studies of polymers in the A conformation, such as transfer RNA and DNA-RNA hybrids, indicated that marked base tilting does not affect the ability of nucleic acids to cause quenching or enhancement of quinacrine fluorescence. 5. Salts inhibit the binding of quinacrine to DNA. 6. Spermine, polylysine and polyarginine, which bind in the small groove of DNA, inhibited quinacrine binding and quenching, while histones, which probably bind in the large groove, had little effect. This correlated with the observation that removal of histones with acid has no effect on Q-banding. 7. Mouse liver chromatin was separated into five fractions. At concentrations of quinacrine from 2×10?6 to 2×10?5 M all fractions inhibited to varying degrees the ability of the chromatin DNA to bind quinacrine and quench quinacrine fluorescence. At saturating levels of quinacrine two fractions, the 400 g pellet (rich in heterochromatin) and a dispersed euchromatin supernatant fraction, showed a decreased number of binding sites for quinacrine. These two fractions were also the richest in non-histone proteins. 8. DNA isolated from the different fractions all showed identical quenching of quinacrine fluorescence. 9. Mouse GC-rich, mid-band, AT-rich, and satellite DNA, isolated by CsCl and Cs2SO4-Ag+ centrifugation all showed identical quenching of quinacrine fluorescence, indicating that within a given organism, except for very AT or GC-rich satellites, the variation in base composition is not adequate to explain Q-banding. — We interpret these results to indicate that: (a) quinacrine binds to chromatin by intercalation of the three planar rings with the large group at position 9 lying in the small groove of DNA, (b) most pale staining regions are due to a decrease binding of quinacrine, and (c) this inhibition of binding is predominately due to non-histone proteins. 相似文献
13.
Comparison between histones FV and F2a2 of chicken erythrocyte. II. Interaction with homologous DNA.
The conformation and stability of artificial complexes between chicken erythrocyte DNA and homologous histones FV and F2a2 was studied by circular dichroism (CD) and thermal denaturation followed by both absorbance and CD measurements. The complexes are made after a stepwise potassium fluoride gradient dialysis without urea and studied at low ionic strength (10-minus 3 M). 1) No structural changes of the DNA can be detected up to r equals 0.2 with FV and r equals 0.6 for F2a2. With FV at higher values of r the CD spectrum is altered, indicating the organization of DNA and histones in some kind of aggregate. 2) The conformation of histone molecules inside the complexes is not related to the ionic strength of the medium but to an effective ionic environment close to 0.1 M. This ionic strength would also correspond to the melting temperature of histone-covered DNA. 3) From the analysis of the absorbance melting profile the length of DNA covered with an histone molecule can be estimated. A good agreement is found between the negative charge of this piece of DNA and the net positive charge of the histone. 4) Since the CD transition at 227 nm occurs before the second absorbance transition at 280 nm, the DNA is stabilized no longer by native histone but partially or fully denatured histones. The helical regions of the histone molecule are not involved in the binding process, which appears to be almost purely coulombian and most likely related to some structural fit between the pattern of negative charges in the DNA helix and that of positive charges along the peptide chain. 相似文献
14.
M I Kovacs 《Analytical biochemistry》1986,154(2):420-423
The effect of digestion methods on the determination of phosphorus in cereal lipids was reinvestigated. Samples were either digested with sulfuric acid or ashed in a muffle furnace at 600 degrees C. The standard deviation and the coefficient of variation were significantly higher for the acid-digested samples. Ashing gave more reliable results, especially when large amounts of lipid material had to be oxidized. 相似文献
15.
Christine A. Bricault Karina Yusim Michael S. Seaman Hyejin Yoon James Theiler Elena E. Giorgi Kshitij Wagh Maxwell Theiler Peter Hraber Jennifer P. Macke Edward F. Kreider Gerald H. Learn Beatrice H. Hahn Johannes F. Scheid James M. Kovacs Jennifer L. Shields Christy L. Lavine Fadi Ghantous Bette Korber 《Cell host & microbe》2019,25(1):59-72.e8
16.
Nasser Fardousi Everton Nunes da Silva Roxanne Kovacs Josephine Borghi Jorge O. M. Barreto Sren Rud Kristensen Juliana Sampaio Helena Eri Shimizu Luciano B. Gomes Letícia Xander Russo Garibaldi D. Gurgel Timothy Powell-Jackson 《PLoS medicine》2022,19(7)
BackgroundPay-for-performance (P4P) programmes to incentivise health providers to improve quality of care have been widely implemented globally. Despite intuitive appeal, evidence on the effectiveness of P4P is mixed, potentially due to differences in how schemes are designed. We exploited municipality variation in the design features of Brazil’s National Programme for Improving Primary Care Access and Quality (PMAQ) to examine whether performance bonuses given to family health team workers were associated with changes in the quality of care and whether the size of bonus mattered.Methods and findingsFor this quasi-experimental study, we used a difference-in-differences approach combined with matching. We compared changes over time in the quality of care delivered by family health teams between (bonus) municipalities that chose to use some or all of the PMAQ money to provide performance-related bonuses to team workers with (nonbonus) municipalities that invested the funds using traditional input-based budgets. The primary outcome was the PMAQ score, a quality of care index on a scale of 0 to 100, based on several hundred indicators (ranging from 598 to 660) of health care delivery. We did one-to-one matching of bonus municipalities to nonbonus municipalities based on baseline demographic and economic characteristics. On the matched sample, we used ordinary least squares regression to estimate the association of any bonus and size of bonus with the prepost change over time (between November 2011 and October 2015) in the PMAQ score. We performed subgroup analyses with respect to the local area income of the family health team. The matched analytical sample comprised 2,346 municipalities (1,173 nonbonus municipalities; 1,173 bonus municipalities), containing 10,275 family health teams that participated in PMAQ from the outset. Bonus municipalities were associated with a 4.6 (95% CI: 2.7 to 6.4; p < 0.001) percentage point increase in the PMAQ score compared with nonbonus municipalities. The association with quality of care increased with the size of bonus: the largest bonus group saw an improvement of 8.2 percentage points (95% CI: 6.2 to 10.2; p < 0.001) compared with the control. The subgroup analysis showed that the observed improvement in performance was most pronounced in the poorest two-fifths of localities. The limitations of the study include the potential for bias from unmeasured time-varying confounding and the fact that the PMAQ score has not been validated as a measure of quality of care.ConclusionsPerformance bonuses to family health team workers compared with traditional input-based budgets were associated with an improvement in the quality of care.Nasser Fardousi and colleagues investigate the association between performance bonuses and the quality of primary health care delivered by family health teams in Brazil. 相似文献
17.
Vasilakis N Falvey D Gangolli SS Coleman J Kowalski J Udem SA Zamb TJ Kovacs GR 《Nature biotechnology》2003,21(8):932-935
This report describes a transfection-independent system for packaging alphavirus replicon vectors using modified vaccinia virus Ankara (MVA) vectors to express all of the RNA components necessary for the production of Venezuelan equine encephalitis (VEE) virus replicon particles (VRP). Infection of mammalian cells with these recombinant MVA vectors resulted in robust expression of VEE structural genes, replication of the alphavirus vector and high titers of VRP. In addition, VRP packaging was achieved in a cell type (fetal rhesus lung) that has been approved for the manufacturing of vaccines destined for human use. 相似文献
18.
K L Kovacs L C Seefeldt G Tigyi C M Doyle L E Mortenson D J Arp 《Journal of bacteriology》1989,171(1):430-435
We examined the immunological cross-reactions of 11 different hydrogenase antigens with 9 different hydrogenase antibodies. Included were antibodies and antigens of both subunits of the hydrogenases of Bradyrhizobium japonicum and Thiocapsa roseopersicina. The results showed a strong relationship among the Ni-Fe dimeric hydrogenases. The two subunits of Ni-Fe dimeric hydrogenases appeared immunologically distinct: specific interactions occurred only when antibodies to the 60- and 30-kilodalton subunits reacted with the 60- and 30-kilodalton-subunit antigens. The interspecies cross-reactions suggested that at least one conserved protein region exists among the large subunits of these enzymes, whereas the small subunits are less conserved. Antibodies to the Fe-only bidirectional hydrogenase of Clostridium pasteurianum reacted with the Desulfovibrio vulgaris bidirectional hydrogenase. Surprisingly, antibodies to the clostridial uptake hydrogenase did not react with any of the Fe-only bidirectional hydrogenases but did react with several of the Ni-Fe dimeric hydrogenases. The two hydrogenases from C. pasteurianum were found to be quite different immunologically. The possible relationship of these findings to the structure and catalytic functions of hydrogenase are discussed. 相似文献
19.
Phospholamban forms Ca2+-selective channels in lipid bilayers 总被引:2,自引:0,他引:2
R J Kovacs M T Nelson H K Simmerman L R Jones 《The Journal of biological chemistry》1988,263(34):18364-18368
Phospholamban is the major membrane protein of the heart phosphorylated in response to beta-adrenergic stimulation. A role for phospholamban in the control of Ca2+ transport by the sarcoplasmic reticulum has been postulated, but the mechanism is incompletely understood. Structural characterization of the purified protein suggests that it is capable of forming a membrane-spanning pore (Simmerman, H. K. B., Collins, J. H., Theibert, J. L., Wegener, A. D., and Jones, L. R. (1986) J. Biol. Chem. 261, 13333-13341). The experiments described here tested the hypothesis that canine cardiac phospholamban, isolated in the fully dephosphorylated state, forms ion channels in lipid bilayers. Phospholamban purified by two different methods formed channels that were permeable to cations, exhibited spontaneous openings and closings, and were selective for Ca2+ over K+. Dihydropyridine drugs and ryanodine did not affect channel activity. The putative membrane-spanning portion of the molecule, residues 26-52, also formed channels in the bilayer. The putative regulatory portion of the molecule, residues 2-25, did not. The results suggest that phospholamban may regulate sarcoplasmic reticulum Ca2+ flux by acting as a Ca2+ channel. 相似文献
20.
Differential effects of tetracaine on charge movements and Ca2+ signals in frog skeletal muscle 总被引:2,自引:5,他引:2 下载免费PDF全文
The effects of tetracaine on charge movements and on antipyrylazo III signals monitoring intracellular delta [Ca2+] were compared in cut frog semitendinosus muscle fibers in a single vaseline gap-voltage clamp. Low tetracaine concentrations (25-40 microM) markedly reduced delta [Ca2+] signals and shifted the rheobase. However, they neither influenced charge movement nor that peak delta [Ca2+] value associated with the contractile threshold. Higher tetracaine concentrations (100-200 microM) partly inhibited charge movements in cut fibers. They separated a steeply voltage-sensitive charge, some of whose features resembled 'q gamma' reported in intact fibers, and whose movement preceded delta [Ca2+] signals at threshold. These findings: (a) directly confirm an earlier suggestion that tetracaine acts on steps in excitation-contraction coupling rather than myofilament activation; (b) show that tetracaine at low concentrations can directly interfere with sarcoplasmic reticular calcium release without modifying charge movement; (c) show that the tetracaine-sensitive charge, first found in intact fibers, also exists in cut fibers; and (d) make it unlikely that tetracaine-sensitive charge transfer is a consequence of Ca2+ release as suggested on earlier occasions. 相似文献