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1.
Sanguinarine, a plant DNA-intercalator, is shown to inhibit the enzyme activity of the membrane-bound Ca2+-ATPase of rabbit skeletal muscle sarcoplasmic reticulum fragments. This inhibition could be interpreted by the well known ability of this alkaloid to interact with sulphydryl groups of the enzymes. Sanguinarine is a weaker inhibitor of this reaction than a sulphydryl group poison Ag+. The I50 is 3.10(-6) M for Ag+ and 7.10(-5) M for sanguinarine in the reaction medium with NO3- substituted for Cl-. In the standard reaction medium containing Cl-, the I50 for sanguinarine is 1.8.10(-4) M. In this case sanguinarine activates Ca2+-ATPase at low concentrations presumably because of uncoupling ATP hydrolysis from Ca2+ transport through membrane. Other agents studied are: DNA-intercalators--ethidium bromide, acriflavine, acridine orange; DNA-complexing antibiotics--actinomycin D, and olivomycin, alkaloids, quinine, morphine, berberine and an uncoupler of oxidative phosphorylation 2,4-dinitrophenol. These were found not to inhibit Ca2+-ATPase activity up to the concentrations of 10(-3)-10(-4) M. 相似文献
2.
A study was made of the dependence of ATP hydrolysis intensity upon different ratios of sodium and potassium ions in plasma membrane of L cells and of cells of clone Lebr 625, sensitive and resistant to ethidium bromide, and of the distribution of cells according to cell cycle phases in dense and sparse cultures. In dense cultures, the cell growth is arrested on G1 phase, the hydrolytic activity of (Na+ + K+)-ATPase decreases, and the Na+, K+ ratio for maximum activity of (Na+ + K+)-ATPase changes. The higher proliferative activity of Lebr 625 cells in dense culture corresponds to the higher hydrolytic activity of (Na+ + K+)-ATPase. 相似文献
3.
Lebr 625 and Lebr 350 cells, resistant to ethidium bromide in concentrations 25 and 50 mkg/ml, are able to grow continuously in serum- and protein-free media. Under the same conditions the parental L929 cells are not able to. Two cell lines (625 sf and 350 sf) were established capable of growing in serum- and protein free media. It is found that ethidium bromide is toxic for resistant cells grown the in serum-free medium. The addition of serum lowers the toxic action of ethidium bromide. A continuous growth of resistant cells in serum-free medium (under nonselective conditions) leads to a decreased level of resistance, which may nevertheless persist for a long period of cultivation (over 2.5 years). 相似文献
4.
350sf and 625sf cells growing in serum free medium secrete transforming growth factors (TGFs) that induce NIH 3T3 indicator cells to form colonies in soft agar. The addition of 2 ng/ml of EGF increases twice the number of colonies of NIH 3T3 indicator cells. The TGFs secreted by 350sf and 625 sf cells do not compete with 125I EGF for binding to EGF receptors on human A-431 cells. The number of EGF receptors on 350 sf and 625 sf and 625 sf cells continuously grown in serum-free medium do not differ from that of EGF receptors on parental Lebr-350 and Lebr-625 cells continuously grown in the presence of 10% serum. These results suggest that TGFs produced by 350 sf and 625 sf cells are not alpha TGF. It is possible that cells secrete beta TGFs of yet unknown type. 相似文献
5.
The protein kinase C (PK C) activity was determined in the cytosolic and membrane fractions of L- and CHO-K1-cells, both sensitive and resistant to ethidium bromide (EB). In the resistant cells (Lebr-25 and Cebr) a decreased enzyme activity was found in addition to alteration of the enzyme elution profile in the membrane preparations purified by DE-52 cellulose column chromatography. Methyltestosterone treated cells had a decreased enzyme activity in nonpurified membrane preparations in Lebr-25 cells, whereas the enzyme quantity in purified preparations remained the same. The decreased PK C activity on membranes correlates with the rapid proliferation of the resistant cells. The differences found between Lebr-25 and Cebr-cell lines in proliferation response to methyltestosterone correspond to the change of PK C developed due to hormone treatment. 相似文献
6.
In HeLa cells two different types of mutants resistant to the cardiac glycoside ouabain (OuaR mutants) or erythrophleum alkaloid cassaine (CasR mutants) have been obtained. One type of mutants resistant to these compounds (designated as group A) are highly resistant (between 50 and 2000-fold) to various cardiac glycosides and their genins such as ouabain, oleandrin, digitoxin, digitoxigenin, strophanthidin, convallatoxin, gitoxin, gitoxigenin, gitaloxin, bufalin, and digoxigenin, but exhibit no cross-resistance to SC4453, a digoxin analog which contains a pyridazine ring in place of the lactone ring in the C-17 position. The second type of mutants (group B) exhibit cross-resistance to all of the cardiac glycosides including SC4453, but their level of resistance is at least 5-10-fold less than that of group A mutants. Interestingly, both groups of mutants exhibited similar degree of cross-resistance towards digoxin and actodigin (AY22241), indicating some differences in their behavior from other cardiac glycosides. Both classes of mutants exhibit no cross-resistance to a wide variety of other structurally and functionally related compounds, e.g. sanguinarine nitrate, ethacrynic acid, penicillic acid, veratridine, harmaline hydrochloride, 5,5'-diphenylhydantoin, quindonium bromide, methyl quinolizinum bromide, estradiol 17 beta-acetate, 21-acetoxy-pregnenolone, vanadium pentoxide, digitonin, and adriamycin, indicating that the genetic lesions in both groups of mutants are specific for cardiac glycosides. This inference is supported by the observation that both group A and B mutants show reduced binding of [3H]ouabain. In group A mutants, a part of the Na+/K+-ATPase activity is highly resistant to inhibition by ouabain, indicating that the genetic lesion in these mutants directly affects Na+/K+-ATPase. In contrast, the Na+/K+-ATPase from the group B mutants showed similar resistance towards ouabain and SC4453 as observed for the parental HeLa cells, indicating that these mutants are affected in a cellular component, other than Na+/K+-ATPase, which is involved in the interaction of cardiac glycosides with the cells. The lack of cross-resistance of the group A mutants to SC4453 and normal sensitivity of their Na+/K+-ATPase to this compound provides strong evidence that the mechanism of interaction of SC4453 with Na+/K+-ATPase differs from that of other cardiac glycosides. 相似文献
7.
Fumaria capreolata was taken into cell suspension culture. The culture yielded a biomass of about 12 g dry weight per liter of medium; the dried cells contained ca. 0.1% of alkaloids. Besides choline, the following ten known isoquinoline alkaloids were isolated from the cell extract in crystalline form: coptisine, dehydrocheilanthifoline; (+)-isoboldine; magnoflorine; N-methylcoclaurine; (+)-reticuline; (–)-pallidine; protopine; sanguinarine; (–)-scoulerine. This is the most diverse isoquinoline alkaloid spectrum thus far published for a cell suspension culture. 相似文献
8.
A study was made of the ouabain effect (10(-3] on cell proliferation and the dependence of ATP hydrolysis on Na/K-concentration in homogenates of mouse hepatoma (XXIIa) and of L-cells, both sensitive and resistant to etidium bromide. Na+, K+-ATPase activity was found in homogenates of cells from sparse cultures in the presence of ouabain, the activity being stimulated by the Na/K-ratio pecular for the maximum enzymatic activity in cells from the dense cultures. The effect of ouabain on the cell proliferation is similar to the effect of transition of sparse cultures to dense ones. 相似文献
9.
Effect of polymeric adsorbents on the production of sanguinarine by Papaver somniferum cell cultures 总被引:2,自引:0,他引:2
The suitability of adsorbent polymeric resins, Amberlite XAD-4 and XAD-7 (Rohm and Hass, Inc.), was investigated for the accumulation of sanguinarine from Papaver somniferum cell cultures. The adsorption and desorption of sanguinarine from aqueous solution was most effective with XAD-7. In addition to sanguinarine, the resins were found to absorb growth regulators and vitamins from the culture medium. Growth inhibition was overcome by delaying for approximately 4 days resin addition after cell inoculation in fresh medium. Resin addition (5% wt/vol) to actively growing uneclicited cultures led to increases in sanguinarine production and release of 30% to 40% and 60%, respectively. The addition of resins to elicited cultures led to increases in alkaloid production of up to 50% to 85% with similar increases in alkaloid release as observed for nonelicited cells. Overall yield of sanguinarine increased from 21 mg . g biomass dry weight(-1) (dw) for elicited cultures to more than 39 mg . gdw(-1) when elicitation was combined with resin addition. Higher quantities of resin (10% to 20% wt/vol) increased marginally the release of sanguinarine into the medium, and on the resin, up to 85% of total production. The use of resin appears promesing for the development of a bioprocess for sanguinarine production by cultured plant cells. (c) 1992 John Wiley & Sons, Inc. 相似文献
10.
Tse WK Chow SC Lai KP Au DW Wong CK 《Journal of experimental zoology. Part A, Ecological genetics and physiology》2011,315(7):385-393
In this study, we aimed to establish an experimental model to study the role of the gill mitochondrion-rich cells (MRCs) of freshwater fish in Na(+) uptake and to examine the effect of adjusting external Na(+) and Cl(-) ions on selected ion transporters in gill MRCs. Japanese eels (Anguilla japonica) acclimated to deionized (DI) water for 2 weeks were transferred directly to (a) ion-supplemented artificial freshwater (AF), (b) Na(+) -deficient AF, or (c) Cl(-) -deficient AF for 2 days. The effects of the transfer on the expression levels of ion transporters in isolated gill cells were investigated. Our data demonstrated that the 2-day acclimation in ion-supplemented AF, Na(+) -deficient AF, or Cl(-) -deficient AF led to a significant increase in serum osmolarity attributed mainly to an increase in serum Na(+) and/or Cl(-) levels when compared with DI-acclimated eel. Significant inductions of V-type H(+) -ATPase (V-H(+) -ATPase) and cotransporter (NBC1) mRNA expression in gill MRCs were detected in AF-acclimated fish. In fish acclimated to Na(+) -deficient AF, mRNA expression levels of V-H(+) -ATPase, NBC1, and Na(+) /H(+) -exchanger-3 (NHE3) were significantly increased in MRCs. Fish acclimated to Cl(-) -deficient AF showed no observable change in expression levels of ion transporters in gill MRCs. In addition, expression levels of ion transporters in pavement cells were stable throughout the 2-day experiments. These data indicate that the level of Na(+) in freshwater is important for altering the mRNA expression of ion transporters in gill MRCs, which supports the notion that gill MRCs play important roles in freshwater Na(+) uptake. 相似文献
11.
The increase of the contour length of the low molecular linear duplex DNA in the complex with an alkaloid sanguinarine has been evidenced by the viscometric method. The enzymatic hydrolysis of modified DNA by pancreatic deoxyribonuclease I and RNA synthesis of DNA by rat liver nuclear RNA polymerase were studied. Sanguinarine has been shown to inhibit the first stages of DNA hydrolysis. This alkaloid is a weaker inhibitor than ethidium bromide, a more potent inhibitor than actinomycin D and exerts an inhibiting effect similar to that of distamycin A. Sanguinarine also decreases the rate of the labelled precursor incorporation into the acid-insoluble fractions by nuclear RNA polymerase from rat liver. A 50% inhibition by sanguinarine was observed at the same alkaloid concentration as that of ethidium bromide. 相似文献
12.
Pelis RM Zydlewski J McCormick SD 《American journal of physiology. Regulatory, integrative and comparative physiology》2001,280(6):R1844-R1852
Na(+)-K(+)-2Cl(-) cotransporter abundance and location was examined in the gills of Atlantic salmon (Salmo salar) during seawater acclimation and smolting. Western blots revealed three bands centered at 285, 160, and 120 kDa. The Na(+)-K(+)-2Cl(-) cotransporter was colocalized with Na(+)-K(+)-ATPase to chloride cells on both the primary filament and secondary lamellae. Parr acclimated to 30 parts per thousand seawater had increased gill Na(+)-K(+)-2Cl(-) cotransporter abundance, large and numerous Na(+)-K(+)-2Cl(-) cotransporter immunoreactive chloride cells on the primary filament, and reduced numbers on the secondary lamellae. Gill Na(+)-K(+)-2Cl(-) cotransporter levels were low in presmolts (February) and increased 3.3-fold in smolts (May), coincident with elevated seawater tolerance. Cotransporter levels decreased below presmolt values in postsmolts in freshwater (June). The size and number of immunoreactive chloride cells on the primary filament increased threefold during smolting and decreased in postsmolts. Gill Na(+)-K(+)-ATPase activity and Na(+)-K(+)-2Cl(-) cotransporter abundance increased in parallel during both seawater acclimation and smolting. These data indicate a direct role of the Na(+)-K(+)-2Cl(-) cotransporter in salt secretion by gill chloride cells of teleost fish. 相似文献
13.
Cultured cells of Eschscholzia californica respond to a yeast glycoprotein elicitor by producing benzophenanthridine alkaloids, which are excreted into the cell wall and the outer medium. These compounds, preferentially sanguinarine, are efficient phytoalexins because of their ability to intercalate double-stranded DNA (dsDNA), penetrate membranes and inhibit various enzymes containing SH-groups. Externally added sanguinarine is rapidly taken up by intact cells and converted to dihydrosanguinarine, which is substituted intracellularly according to the biosynthetic route. A 29.5 kDa soluble enzyme that catalyses the reduction of sanguinarine and chelerythrine by either NADPH or NADH has been isolated and purified to homogeneity. Benzophenanthridines that accumulate in the outer medium, mainly 10-OH-chelerythrine, chelirubine and macarpine, are converted by the isolated enzyme and by intact cells at much slower rates than sanguinarine. The cellular capacity of uptake and conversion of sanguinarine largely surpasses the rate of alkaloid production. We conclude that the sanguinarine produced by intact cells, after excretion and binding to cell wall elements, is rapidly reabsorbed and reduced to the less toxic dihydrosanguinarine, which then undergoes further biosynthetic reactions. This recycling process would allow the presence of the toxic phytoalexin at the cellular surface without taking the risk of injuring the producing cell. 相似文献
14.
Effects of some metabolic inhibitors, as well as of biologically active compounds (diakarb, ethidium bromide and a phenanthridine alkaloid sanguinarine) on the formed novocaine and neutral red segregation zones were studied. The volume of granules diminished under the influence of a glycolytic inhibitor iodoacetate, uncouplers of oxidative phosphorylation (2,4-dinitrophenol and carbonyl cyanide trifluoromethoxyphenylhydrozone), and respiratory inhibitors (antimycin A and rotenone), as well as under the influence of cycloheximide - an inhibitor of protein synthesis. Diakarb, ethidium bromide or sanguinarine also provoked a regression of the segregation zones. It has been found that all these compounds are inhibitors of ATPase activity of the isolated segregation zones. A possible mechanism of volume decreasing in segregation zones under the influence of both the metabolic inhibitors and diakarb, ethidium bromide and sanguinarine is discussed. 相似文献
15.
Using L-cells both sensitive and resistant to cytotoxic action of ethidium bromide (EB), a study was made of the intracellular level of cAMP, activities of adenylcyclase, phosphodiesterase and cAMP, liberated from cells into the surrounding medium. In EB resistant L-cells compared to EB sensitive ones, the higher level of cAMP with a decreased activity of adenylcyclase and an increased activity of the phosphodiesterase was shown to be associated with an impeded exit of cAMP from cells. It is suggested that the differences in cAMP levels in the EB sensitive and resistant cells are associated with the properties of cAMP-dependent protein kinases of these cells. 相似文献
16.
Na(+),K(+)-ATPase, a basolateral transporter responsible for tubular reabsorption of Na(+) and for providing the driving force for vectorial transport of various solutes and ions, can also act as a signal transducer in response to the interaction with steroid hormones. At nanomolar concentrations ouabain binding to Na(+),K(+)-ATPase activates a signaling cascade that ultimately regulates several membrane transporters including Na(+),K(+)-ATPase. The present study evaluated the long-term effect of ouabain on Na(+),K(+)-ATPase activity (Na(+) transepithelial flux) and expression in opossum kidney (OK) cells with low (40) and high (80) number of passages in culture, which are known to overexpress Na(+),K(+)-ATPase (Silva et al., 2006, J Membr Biol 212, 163-175). Activation of a signal cascade was evaluated by quantification of ERK1/2 phosphorylation by Western blot. Na(+),K(+)-ATPase activity was determined by electrophysiological techniques and expression by Western blot. Incubation of cells with ouabain induced activation of ERK1/2. Long-term incubation with ouabain induced an increase in Na(+) transepithelial flux and Na(+),K(+)-ATPase expression only in OK cells with 80 passages in culture. This increase was prevented by incubation with inhibitors of MEK1/2 and PI-3K. In conclusion, ouabain-activated signaling cascade mediated by both MEK1/2 and PI-3K is responsible for long-term regulation of Na(+) transepithelial flux in epithelial renal cells. OK cell line with high number of passages is suggested to constitute a particular useful model for the understanding of ouabain-mediated regulation of Na(+) transport. 相似文献
17.
The gene encoding vacuolar H(+)-ATPase subunit C is overexpressed in multidrug-resistant HL60 cells.
Previous studies have suggested that vacuolar H(+)-ATPase activity may play a role in modulating drug transport mechanism in multidrug resistant HL60 cells. In the present study we have used a cDNA of human vacuolar H(+)-ATPase subunit C (SC-H(+)-ATPase) to analyze expression of this gene in HL60 cells isolated for resistance to adriamycin or vincristine. The results demonstrate that development of resistance to either agent results in a major increase in the levels of SC-H(+)-ATPase mRNA. Furthermore in resistant cells which have partially reverted to drug sensitivity there is a parallel reduction in SC-H(+)-ATPase mRNA levels. Southern blot analysis shows that the SC-H(+)-ATPase gene is not amplified in the resistant cells. These results therefore demonstrate a correlation between the development of multidrug resistance and enhanced expression of the SC-H(+)-ATPase gene. 相似文献
18.
Molecular cloning of the plasma membrane H(+)-ATPase from Kluyveromyces lactis: a single nucleotide substitution in the gene confers ethidium bromide resistance and deficiency in K+ uptake.
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A Kluyveromyces lactis strain resistant to ethidium bromide and deficient in potassium uptake was isolated. Studies on the proton-pumping activity of the mutant strain showed that a decreased H(+)-ATPase specific activity was responsible for the observed phenotypes. The putative K. lactis PMA1 gene encoding the plasma membrane H(+)-ATPase was cloned by its ability to relieve the potassium transport defect of this mutant and by reversing its resistance to ethidium bromide. Its deduced amino acid sequence predicts a protein 899 residues long that is structurally colinear in its full length to H(+)-ATPases cloned from different yeasts, except for the presence of a variable N-terminal domain. By PCR-mediated amplification, we identified a transition from G to A that rendered the substitution of the fully conserved methionine at position 699 by isoleucine. We attribute to this amino acid change the low capacity of the mutant H(+)-ATPase to pump out protons. 相似文献
19.
The mechanisms of cadmium (Cd)-dependent nephrotoxicity were studied in a rat proximal tubule (PT) cell line. CdCl(2) (5 microM) increased the production of reactive oxygen species (ROS), as determined by oxidation of dihydrorhodamine 123 to fluorescent rhodamine 123. The levels of ubiquitin-conjugated cellular proteins were increased by Cd in a time-dependent fashion (maximum at 24-48 h). This was prevented by coincubation with the thiol antioxidant N-acetylcysteine (NAC, 15 mM). Cd also increased apoptosis (controls: 2.4+/-1.6%; Cd: 8.1+/-1.9%), but not necrosis (controls: 0.5 +/- 0.3%; Cd: 1.4+/- 2.5%). Exposure of PT cells with Cd decreased protein levels of the catalytic subunit (alpha1) of Na+/K(+)-ATPase, a long-lived membrane protein (t(1/2)>48 h) that drives reabsorption of ions and nutrients through Na(+)-dependent transporters in PT. Incubation of PT cells for 48 h with Cd decreased Na+/K(+)-ATPase alpha1-subunit, as determined by immunoblotting, by approximately 50%, and NAC largely prevented this effect. Inhibitors of the proteasome such as MG-132 (20 microM) or lactacystin (10 microM), as well as lysosomotropic weak bases such as chloroquine (0.2 mM) or NH(4)Cl (30 mM), significantly reduced the decrease of Na(+)/K(+)-ATPase alpha1-subunit induced by Cd, and in combination abolished the effect of Cd on Na+/K(+)-ATPase. Immunofluorescence labeling of Na+/K(+)-ATPase showed a reduced expression of the protein in the plasma membrane of Cd-exposed cells. After addition of lactacystin and chloroquine to Cd-exposed PT cells, immunoreactive material accumulated into intracellular vesicles. The data indicate that micromolar concentrations of Cd can increase ROS production and exert a toxic effect on PT cells. Oxidative damage increases the degradation of Na+/K(+)-ATPase through both the proteasomal and endo-/lysosomal proteolytic pathways. Degradation of oxidatively damaged Na+/K(+)-ATPase may contribute to the 'Fanconi syndrome'-like Na(+)-dependent transport defects associated with Cd-nephrotoxicity. 相似文献
20.
Marshall WS 《The Journal of experimental zoology》2002,293(3):264-283
The secondary active Cl(-) secretion in seawater (SW) teleost fish gills and elasmobranch rectal gland involves basolateral Na(+),K(+)-ATPase and NKCC, apical membrane CFTR anion channels, and a paracellular Na(+)-selective conductance. In freshwater (FW) teleost gill, the mechanism of NaCl uptake is more controversial and involves apical V-type H(+)-ATPase linked to an apical Na(+) channel, apical Cl(-)-HCO-3 exchange and basolateral Na(+),K(+)-ATPase. Ca(2+) uptake (in FW and SW) is via Ca(2+) channels in the apical membrane and Ca(2+)-ATPase in the basolateral membrane. Mainly this transport occurs in mitochondria rich (MR) chloride cells, but there is a role for the pavement cells also. Future research will likely expand in two major directions, molded by methodology: first in physiological genomics of all the transporters, including their expression, trafficking, operation, and regulation at the molecular level, and second in biotelemetry to examine multivariable components in behavioral physiological ecology, thus widening the integration of physiology from the molecular to the environmental levels while deepening understanding at all levels. 相似文献