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Printed in the Netherlands. Topoisomerase Home work caserta assembly is home work caserta assembly nuclear enzyme localized at active sites of transcription, and abnormal levels of the enzyme have been observed in a variety of neoplasms.
Because the enzyme binds heparin and, given the presence of heparan sulfate within the nuclei of mammalian cells, we sought to investigate the interaction between topoisomerase I and sulfated glycosaminoglycans isolated from normal and neoplastic human liver.
The results demonstrated that low concentrations ~ nM of heparan sulfate from normal liver but not from its malignant counterpart effectively blocked relaxation of supercoiled DNA driven by either purified holoenzyme or topoisomerase I activity present in nuclear extracts of three malignant cell lines.
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Heparin acted at even lower ~10 nM concentrations. Our results suggest that DNA topoisomerase I activity may be modulated in vivo by speci; fic heparan sulfate moieties present in normal cells but markedly reduced home work caserta assembly absent in their transformed counterparts. Eukaryotic topoisomerase I topo-I is a nuclear enzyme implicated in the relaxation of torsionally-strained duplex DNA, and thus capable of relaxing both negatively and positively supercoiled DNA [1, 2].
Bár jóval kevésbé angolos és nyersebb electro, mint elődje, annyira azért nem lett szerencsére amerikanizált a hangzás, de akad rá példa: a kilencvenes évek amerikai bojgruppjainak hangzását idéző No ID, amire a klip is rátesz pár lapáttal. Ennek ellenére egy kellemes darabról van szó. Az első album telitalálat lett, köszönhetően az egyedi hangzásvilágának, Frank hangjának és a lemezen megbúvó mélységeknek, viszont a Do It In The AM-en sajnos nem csak a hangi adottságait nem használja ki Frank, de a mélységek is hiányoznak belőle. Ballada nem található rajta pedig Frank erős bennükhelyettük csak és kizárólag bulizós darabok vannak, és ezek közt is csak néhány ér fel a bemutatkozó album ütős electro bombáihoz. A lemez maradéka is szinte végig jó dalokat tartalmaz, amik közül éterium felülvizsgálata kiemelkedő a Footsteps, a Struck By Lightning, a 80as éveket idéző The Fear Inside ez pedig még két éve íródott, ami meg is hallatszik rajta, mert eléggé más, mint a lemez egésze.
To fulfill this function, topo-I introduces single strand nicks into the DNA allowing the intact strand to pass through the nick; subsequently, the nick is rejoined by the enzyme. Topo-I is essential for transcription [3—5] and is known to be highly concentrated in regions of the chromatin that are undergoing active transcription such as activated Address for offprints: R.
Furthermore, transcription can be inhibited by anti-topo-I antibodies [8]. A fundamental question is whether topo-I is required for DNA replication. Although home work caserta assembly topo-I and II are required for chain elongation [2], only topo-I is responsible for the swivel function home work caserta assembly replication of mammalian cells [9].
The role of topo-I in gene transcription and DNA replication suggests that this enzyme may also play a role in tumor development. Indeed, increased topo-I activity has been detected in a variety of human malignancies, and has been utilized as a molecular target for antitumor therapy [10—13].
In addition, altered topo-I enzymatic activity and abnormal structure of the holoenzyme has been associated with the development of drug resistance [14—16]. In spite of this body of evidence on topo-I function, little is known about the factors that amit a trendvonal egyenlete mutat regulate its activity in vivo.
The enzyme is now considered to be regulated by home work caserta assembly activity of protein kinase C or casein kinase II-like protein kinases [20, 21]. In agreement with this view, is the fact that inhibitors of either tyrosine kinase [22] or poly-ADP-ribosylation [23] can effectively block Topo-I enzyme function. Indeed, a recent study has shown that mammalian topo-I has phosphokinase activity and can phosphorylate specific serine residues of SR proteins [24].
Heparin, a highly-sulfated glycosaminoglycan GAGcan inhibit topo-I activity in vitro [25] and its heparin-binding ability has been utilized to isolate and purify prokaryotic enzyme in large quantities [26]. Home work caserta assembly great deal of evidence has been accumulated indicating that sulfated GAGs, primarily heparan sulfate, are present, at least transiently, in the nuclei of mammalian cells [27—29], and their implication in growth control has also been suggested [30].
Of note, targeting of specific heparan sulfate sequences to the nuclei of hepatoma cells augments with growth suppression and declines with growth stimulation [27, 28]. Moreover, exogenous heparan sulfate can reach the nucleus and exert similar inhibitory effects [31]. In the present study, we sought to investigate the interaction between topo-I and GAGs purified from normal or neoplastic liver tissue.
Because it is well established that the effects of GAGs strongly depend on their fine molecular architecture [31, 34—37], we hypothesized that structural alterations of GAGs detected in hepatocellular carcinoma [38] might modify their inhibitory action on topo-I activity, thereby favoring tumor progression. This possibility was tested in a cell-free system by using purified holoenzyme or nuclear extracts from either normal or neoplastic liver as well as established cell lines. Our results demonstrate that micromolar concentrations of heparan sulfate from normal liver but not from hepatocellular carcinoma are capable of blocking topo-I activity.
Moreover we show that basic fibroblast growth factor bFGF interferes with this inhibitory activity and that both heparin-binding sites and bFGF immunoreactive loci co-localize in the nuclei of U leukemic cells. We propose that topo-I enzyme activity may be modulated in vivo by specific heparan sulfate moieties present in normal cells but markedly reduced or absent in their transformed home work caserta assembly.
Materials and methods Chemicals and materials All chemicals for buffers and electrophoretic purposes were purchased from Merck Germany and Sigma, respectively. Ultrapure agarose was the product of BRL.
Heparan sulfate from bovine kidney, dermatan sulfate from porcine skin, chondroitin 4-S and 6-S from shark cartilage, and chondroitinase ABC were obtained from Seikagaku Japan. Heparin from porcine intestinal mucosa, with average Mr of 11 kDa, was bought from Serva Germany. Biotin-labelled GAGs and dextran sulfate [39] were generously provided by Dr. Gabius Germany. Purified human placenta topo-I and rabbit polyclonal antibody against the enzyme were obtained from TopoGen Inc.
Human liver specimens, surgically removed from organ donors essentially the portions that were not utilized for transplantation into pediatric patients or following partial hepatectomy because of hepatocellular carcinoma, were immediately frozen in liquid nitrogen and stored at —70°C until analyzed.
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The dry powder was dissolved in 0. The yield of total GAGs was determined by measuring the uronic acid content of the samples [42]. To isolate heparan sulfate, the samples were extensively digested with chondroitinase ABC as previously described [38]. The size of the heparan sulfate and total GAG chains determined by gradient polyacrylamide gel electrophoresis centered at ~15 kDa not shown.
This allowed calculations of GAG molarities. To establish purity of the various GAG preparations, digested and undigested samples were analyzed by cellulose acetate electrophoresis.
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The electrophoretic characteristics of normal and neoplastic heparan sulfate were identical in all preparations. Samples were incubated at 37°C for 30 min with or without various GAGs as indicated. The amount of GAGs varied between 1 and ng in the reaction mixtures, and these concentrations did not influence the pH. Gel was post-stained with 0. Tests for nuclease contamination were performed by assaying generation of linearized plasmid DNA using incubation buffers and times home work caserta assembly above.
No linear DNA or breakdown products were detected.
The relative density of supercoiled plasmid was compared to the density of relaxed forms. All data represent the average of 4 independent experiments ± S.
Cells were grown in Luria-Bertani medium overnight. Nuclear extracts, topo-I relaxation assay and quantitation of topo-I activity Nuclei were isolated from U, HT and HT cells according to a previous method [44] including various protease inhibitors.
Samples were further extracted with 0. Protein concentrations were determined by a colorimetric assay [41]. ATP-independent relaxation of supercoiled pBluescript plasmid was done in a standard 30 µl reaction mixture containing either 2 U ~2 ng placenta topo-I enzyme 1 U of enzyme can relax 0.
Parallel aliquots from the same extracts were used to prepare immuno-dot blot. The specific reactions were then visualized with anti-goat secondary antibody labelled with alkaline phosphatase.
Antibody against topo-I was used in dilution and visualized by peroxidase-labelled protein A 1: After a subsequent blocking step, the cells were reacted with polyclonal goat anti-bFGF antiserum and visualized by rhodamine-labelled home work caserta assembly anti-goat antibody. Controls with or without strepatvidin-FITC or lacking the primary antibodies were run in parallel. The specific fluorescence patterns were visualized by MRC confocal laser scanning system Biorad.
All the reaction mixtures contained 2 U of purified enzyme ~2 ng and 0. The results showed that heparan sulfate, dermatan sulfate or chondroitin sulfate from commercial sources see Materials and methods did not have any effect on topo-I activity Figs 1A and 1B, lanes 2—4. In contrast, 1 µg of total GAGs isolated from either normal lane 5 or neoplastic lane 7 liver, home work caserta assembly 0.
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Of note, 0. These results were confirmed in 4 independent experiments with minimal variations. Effects of liver glycosaminoglycans and heparan sulfate on topoisomerase I activity in nuclear extracts from neoplastic cells To estimate the role of other nuclear factors in the GAGinduced topo-I inhibition, 0. Total liver GAGs 1 µgwhich contained primarily heparan sulfate as the active inhibitory fraction on the human placenta holoenzyme, opciós prémium kerül meghatározásra used in the plasmid relaxation assay.
The results Fig. However no effect was observed in the nuclear extracts isolated from U monocytic leukemia cells Fig. To address these possibilities, we performed an additional purification step by subjecting the 0. Inhibitory action of glycosaminoglycans on purified topo-I activity. The electrophoretic picture A was reproduced in four independent experiments and the quantitative data obtained by scanning densitometry are shown in the bar graph B.
The values are the mean ± S. The numbers at the bottom of panels A and B correspond to each other and include: 2 U of topo-I holoenzyme alone lane 1 or in the presence of 1 µg heparan sulfate from bovine kidney lane 21 µg of dermatan sulfate from porcine skin lane 31 µg of chondroitin 4-sulfate from shark cartilage lane 41 µg of total GAGs from normal liver lane 50.
These results, opciós kereskedési rendszer, indicate that the levels of topo-I home work caserta assembly U nuclei were not abnormally elevated, a fact that might have accounted for the lack of inhibition by liver heparan sulfate. Instead, the data are consistent with the presence of an activity blocking the GAG-induced inhibition of topo-I.
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Of note, inhibition of topo-I, albeit to a lesser degree, was home work caserta assembly obtained in the presence of total liver GAGs, liver heparan sulfate and total GAGs isolated from hepatocellular carcinoma Fig. However, heparan sulfate from liver carcinoma exerted no inhibitory effect Fig.
Taken together, the results presented above indicate that a normal liver heparan sulfate can modulate topo-I activity both on purified placenta holoenzyme and on nuclear enzyme preparations, before or after PEG precipitation, from three histogenetically distinct tumor cell lines; and b a PEG-precipitable factor can interfere with this heparan sulfate-driven inhibition.
A GAG-binding nuclear protein is responsible for blocking the inhibitory ability of heparan sulfate on topoisomerase I activity Fig. Inhibitory action of total glycosaminoglycans from normal liver on nuclear extracts of various human tumor cell lines.
The values represent the mean ± S. The experiments outlined above raised the possibility that a nuclear factor could have interfered with heparan sulfate action on topo-I activity. Therefore, we sought to investigate the nature of this factor.
In initial experiments, the rapid disappearance of the inhibitory activity from U samples that were not carefully protected against protease not shown suggested the involvement of a protein in this Fig. Effects of normal liver glycosaminoglycans on the topo-I activity of native and PEG-precipitated nuclear extracts from U cells.
The data were quantified by scanning laser densitometry and computer integration. Notice that the same results were obtained when liver GAGs isolated from a different home work caserta assembly were used lanes 5 and 6. Inhibitory action of normal and neoplastic liver glycosaminoglycans or heparan sulfate HS on topo-I activity of PEG-precipitated U nuclear extracts.
Results are expressed as percent of control filled bar and represent the average ± S. We noted that, although the majority of proteins were retained in the PEG soluble fraction Fig. To dem- onstrate GAG binding capacity of the proteins in these fractions, the PEG precipitate, its supernatant, and the original nuclear home work caserta assembly were immobilized onto nylon membrane and reacted with various biotin-labelled GAGs Fig.
The fractions of the 0. Electrophoretic separation left panel and interaction with various GAGs right panel of nuclear proteins extracted from U tumor cells. The left panel shows a picture of an electrophoretic separation of 40 µg 0. The numbers on the left margin indicate the migration of protein standards. The gel was stained with Coomassie brilliant blue. Notice that the major fraction precipitated by PEG contains a 14—17 kDa protein.
These three fractions were blotted using a dot blot apparatus and reacted with biotin labelled GAGs, including dermatan sulfate DSchondroitin 6-sulfate C6Schondroitin 4-sulfate C4Sheparin HP or dextran sulfate DxS as indicated in the right panel.
The reactions were visualized by streptavidin peroxidase. The pénzt keresni az elektronikus számlájára a, home work caserta assembly, c are the same as in the left panel.
Further confirmation that PEG-precipitable protein could compete with heparan sulfate inhibitory activity was obtained when this material was tested on purified placenta holoenzyme.
Of note, PEG precipitate itself did not influence topo-I activity, but completely abolished the inhibitory action of heparan sulfate present in liver GAGs Fig. To investigate further this phenomenon, the effects of increasing concentrations of exogenous heparin, an homologue of heparan sulfate and a very potent topo-I inhibitor, and of liver heparan sulfate were measured in the presence or absence of PEG-precipitable proteins, that is, GAG-binding proteins GBPs Fig. These data, thus, indicate that 0.
Because the average Mr of isolated Fig. PEG-precipitable nuclear factor abolishes the effect of liver glycosaminoglycans on topo-I activity. Two units of purified topo-I were reacted with 1.
The values are the averages ± S. The slope of the curve in Fig. Nuclear extracts of U cells were used with or without PEG precipitation. Note also that heparan sulfate is also capable of inhibiting the topo-I activity, albeit at higher dosages IC50 ~1. The values are the averages of 4 independent experiments with S. Immunoblotting analyses of the 0. These results, thus, validate the experiments reported above and demonstrate that topo-I is not affected by PEG precipitation.
Moreover, these results suggest that biotinilated heparin bound topo-I enzyme Fig. Of note, both biotinylated heparin Fig. This suggests that other small-Mr proteins, likely histones, may account for some of the immunoreactive material. The overall effects were comparable to those obtained when PEG precipitate alone was used Fig.
