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1.
Immunoglobulin-synthesizing cells from the spleen and bursa were fractionated by the 1 X G sedimentation velocity technique and characterized by their ability to synthesize immunoglobulin and by staining with fluorescent anti-light chain chain. Four subpopulations of immunoglobulin-synthesizing cells were identified. In the bursa, slowly sedimenting (S 2.3 mm/hr) and rapidly sedimenting (S greater than 3.5 mm/hr) subpopulations with surface immunoglobulin were present; in the spleen, a slowly sedimenting (S 2.3 mm/hr) subpopulation with surface immunoglobulin and plasma cells (S greater than 3.5 mm/hr) with large concentrations of intracellular immunoglobulin existed. The subpopulations differed most markedly in their ability to synthesize immunoglobulin (cpm Ig synthesized/10(6) Ig-positive cells); the rates of immunoglobulin synthesis were in the ratio 1:2:1:900. The slowly sedimenting B cells from the spleen and both subpopulations of B cells from the bursa released small amounts of immunoglobulin into the culture media, whereas, the plasma cells released immunoglobulin at a rate as much as 3700 times greater. Bursal B cells could be further distinguished from splenic B cells by a greater amount of DNA synthesis.  相似文献   

2.
In full-grown oocytes of Xenopus laevis more than 80 % of the total DNA polymerase activity is found in the germinal vesicle (nucleus) and only about 8% in the cytoplasm. The intracellular distribution of the multiple DNA polymerase forms has been studied in oocytes and in embryonic cells. The oocyte nucleus contains a major DNA polymerase species, sedimenting at about 7S, and a minor species sedimenting at about 5S. These enzymes are comparable, respectively, with the DNA polymerases α and β described in other biological systems. In the oocyte cytoplasm, besides a small amount of the 7S form, an 8–9S DNA polymerase activity is also detectable. In the nuclei of embryonic cells, in addition to the DNA polymerase forms present in the oocyte nucleus, a new major form which seems specific for the eggs and embryos is detectable by DEAE chromatography.  相似文献   

3.
Abstract: The number of catalytic subunits of purified bovine nucleus caudatus acetylcholinesterase (E.C. 3.1.1.7) has been determined by active site labelling with [3H]diisopropyl fluorophosphate ([3H]DFP). The 10.5 S, 16 S, and 20 S forms were estimated to contain two, four, and six active sites, respectively, per molecule. A 4.8 S form, which showed a weak amphiphile-dependent activity behavior, was obtained by selective proteolytic digestion with pronase. The inability of the purified 4.8 S form to aggregate after detergent removal, and the molecular mass in the range of 130-165 kD under nondenaturating conditions, indicate that this form is a dimeric form, lacking those hydrophobic regions responsible for aggregation.  相似文献   

4.
J P Sine  J P Toutant  P Weigel  B Colas 《Biochemistry》1992,31(44):10893-10900
The properties of a cholinesterase from mucosal cells of rat intestine have been characterized. The enzyme was identified as butyrylcholinesterase because it was more sensitive to iso-OMPA (IC50 = 1.0 x 10(-6) M) than to BW284C51 (IC50 = 5.5 x 10(-5) M) and was not inhibited by substrate excess. It displayed a higher affinity for acetylthiocholine than for butyrylthiocholine. A major molecular form was observed sedimenting at 5.9 S. Two other minor molecular forms were identified as a hydrophilic tetramer (G4, sedimenting at 10.5 S) and a monomer (G1, sedimenting at 4.3 S). The 5.9 S component was referred to as "G" form (G for globular) and not "G2" as usual dimers for the following reasons: (i) the G form was unaffected by the reducing agents, beta-mercaptoethanol and dithiothreitol, which converted disulfide-linked dimers of acetylcholinesterase into monomers, (ii) the G form was shifted from 5.9 to 3.4 S when the sucrose gradient contained Triton X-100. This value of 3.4 S (in Triton X-100) appeared too low for a typical G2 form. The shift in the S value was partly reversible: the 3.4 S form resedimented at 5.2 S in the absence of detergent. The behavior of the G form in sucrose gradients indicated that it was amphiphilic. This was confirmed in nondenaturing electrophoreses and also by quantitative binding of the G form to octyl-Sepharose. The hydrophobic domain of the G form was not a glycolipid, as shown by its insensitivity to Bacillus thuringiensis phosphatidylinositol-specific phospholipase C and its nonaggregating properties in the absence of nondenaturing detergent.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

5.
Over 80% of the phenylalanyl-tRNA synthetase activity in Ehrlich ascites cell homogenates was found to be associated with the high speed particulate fraction. This enzyme activity occurred in two principle forms: activity bound to the ribosomes, and activity as part of a complex sedimenting at approximately 25S in a sucrose density gradient. The ribosome-associated enzyme was shown to be bound to the 60S ribosomal subunit. Exposure of the ribosomes to RNA resulted in removal of synthetase activity from the ribosomes and the concomitant appearance of activity in a complex sedimenting at 25S.  相似文献   

6.
A virus obtained from naturally infected lucerne ( Medicago sativa ) in New Zealand reacted with antiserum to an Australian isolate of lucerne transient streak virus (LTSV). Some plants infected with New Zealand isolates showed yellow flecks along lateral veins of leaves; symptoms were transient in some lucerne plants but persistent in others. A New Zealand isolate (LTSV-NZ) infected 14 of 39 plant species tested by mechanical inoculation, but was not transmitted by five aphid species. In sap of Nicotiana clevelandii , LTSV-NZ was infective after storage for 4 wk at 20 oC, diluting to 10-5, or heating for 10 min at 70 oC. Purified virus preparations contained a single electrophoretic component and a single sedimenting component (s20w= 112 S ) which formed a single buoyant density component in CsCl (1.37 g cm-3) but two density components in Cs2SO4 (1.26 and 1.32 g cm-3). LTSV-NZ particles were stable in 10 ITIM EDTA at pH 5, but not at pH 8, being degraded into two sedimenting components of 105 S and 92 S. Particles contained c. 18% RNA in the form of one single-stranded RNA molecule of mol. wt 1–4 times 106, and a polypeptide of mol. wt c. 32 400. LTSV-NZ was serologically unrelated to 24 other isometric plant viruses. However, its properties are similar to those of southern bean mosaic virus and allied viruses. The present cryptogram of LTSV is R/l: 1–4/(18):S/S:S/*.  相似文献   

7.
In order to investigate the functional organization of the progesterone receptor in chromatin we characterized the physical-chemical properties of the receptor bound chromatin fragments released by micrococcal nuclease and DNase I digestion. The crude nuclear fraction was isolated from T 47 D cells, previously exposed to 0.1 microM [3H]ORG 2058. The parameters determined in low and high salt concentrated buffers were: sedimentation coefficients (S) on a sucrose gradient, Stokes radii (Rs) by gel filtration on a Sephadex G-200 column and the binding abilities to a DNA-cellulose column. The molecular weights (Mr) and frictional ratios (f/fo) were calculated from the S and Rs values. Micrococcal nuclease digestion solubilized a receptor form sedimenting as a single peak at 4.4 S with a Rs = 7.78 nm and an estimated Mr = 144,000. About 53% of the applied receptor bound to a DNA-cellulose column could be eluted by high salt concentrated buffer. 0.4 M KCl dissociated this receptor form into a smaller receptor sedimenting at 3.3 S with Rs = 5.53 nm and a calculated Mr = 76,000. A similar receptor form was extracted by 0.6 M KCl from the undigested crude nuclear fraction. DNase I digestion solubilized a receptor form sedimenting at 3.3 S with a Rs = 6.87 nm and a calculated Mr = 94,000. About 26% of the applied receptor bound to a DNA-cellulose column could be eluted by high salt concentrated buffer. Dissociation of this receptor form by 0.4 M KCl resulted in a receptor sedimenting at 2.8 S with a Rs = 6.53 nm and an estimated Mr = 76,000. These results suggest: The progesterone receptor in chromatin is associated with several molecules probably proteins which complexed it to DNA. Some of these molecules still associated with the progesterone receptor could be released by nucleases digestion. Micrococcal nuclease releases a larger portion of these molecules than those release by DNase I.  相似文献   

8.
Multiple forms of the vesicular stomatitis virus nucleocapsid protein N have been detected in infected cells. One form is complexed with the viral NS protein in a 1:1 molar ratio, and the other forms are distinguished by their more rapid sedimentation rates on glycerol gradients. I performed a series of experiments designed to analyze the relationships between these forms of the N protein. Pulse-chase experiments demonstrate that the N protein is made first as the form which binds to the NS protein, forming a 1-to-1 molar complex, and that with increasing times of chase it is either assembled into nucleocapsids or converted to the two higher sedimenting forms. Using a newly developed quantitative immunoblotting procedure, I have quantitated the three differentially sedimenting species of the N protein and have shown that at later times postinfection (6 to 7 h), the faster-sedimenting forms of the N protein account for as much as 50% of the soluble N protein in the cell. The activity of these forms has been assessed, with only the 1-to-1 molar N-NS complex demonstrating the ability to support the replication and encapsidation of viral genomic RNA. A model for the conversion of the N protein from the active N-NS complex into the other forms of the protein is presented, and the possible function of the N-protein self-complexes is discussed.  相似文献   

9.
Three Size-Classes of Intracellular Adenovirus Deoxyribonucleic Acid   总被引:18,自引:15,他引:3       下载免费PDF全文
When human adenovirus type 2 or 12 infects cells, either productively or non-productively, three classes of viral deoxyribonucleic acid (DNA) are found within the cells: (i) viral DNA which cosediments with DNA extracted from infectious adenovirions at 31.3S for adenovirus type 2 and at 29.0S for adenovirus type 12, (ii) viral DNA which sediments at about 18S, and (iii) viral DNA which sediments at >45S and is apparently integrated into the cellular DNA. A precursor-product relationship is suggested as a working hypothesis; the intact viral DNA is hydrolyzed to slowly sedimenting DNA and the slowly sedimenting DNA is integrated into the cellular DNA. Both the parental and the newly synthesized viral DNA are altered by this route. The intact viral DNA within the cells apparently is cleaved into the slowly sedimenting DNA by a preformed enzyme.  相似文献   

10.
Dopamine beta-hydroxylase exists as three forms in human neuroblastoma (SH-SY5Y) cells. The membrane-bound form of the hydroxylase contains three different species with apparent relative molecular weights of 73,000, 77,000, and 82,000. The intracellular soluble form of dopamine beta-hydroxylase was present as a single species with an apparent molecular weight of 73,000. Pulse-chase experiments showed that membranous dopamine beta-hydroxylase contains two subunit forms of 73,000 and 77,000 after short chase times. The soluble hydroxylase was synthesized as a single species of 73,000 at approximately the same rate as the lower molecular weight species of the membranous enzyme. A constitutively secreted third form of the enzyme with an intermediate apparent molecular weight also incorporated [35S]sulfate, whereas no significant amount of [35S]sulfate was observed in the cellular forms of the enzyme. The [35S]sulfate was incorporated on N-linked oligosaccharides. Approximately 12% of the enzyme is released constitutively within 1 h. These results demonstrate that neuronal cells have the ability to constitutively secrete a specific form of dopamine beta-hydroxylase which may contribute to the levels of this enzyme found in plasma.  相似文献   

11.
In various permissive monkey cell lines infected with simian virus 40 there are two major forms of large T antigen which differ in their rate of sedimentation through sucrose gradients. The lighter (5 to 7S) form sedimented slightly more rapidly than the 4S tRNA marker, whereas the heavier (16S) form sedimented slightly more slowly than the 18S rRNA marker. The small t antigen did not form complexes which sedimented as rapidly as those formed by the large T antigen. The 16S T antigen form was converted to the slowly sedimenting 5 to 7S form in the presence of 1.0 M NaCl. The majority of large T antigen synthesized in cell-free protein-synthesizing systems primed by mRNA isolated from infected cells sedimented as the 5 to 7S form even when premixed with excess quantities of cellular T antigen. The formation of the 16S form in infected cells did not require ongoing viral or cellular DNA replication because considerable quantities of this T antigen class were produced in the presence of DNA synthesis inhibitors, such as cytosine arabinoside. Both 5 to 7S and 16S forms could be isolated separately and, therefore, each could be analyzed as to its individual properties. The 5 to 7S T antigen form bound more efficiently and tightly to DNA and had specific affinity for sequences at the viral origin of replication, whereas the 16S form bound less efficiently to DNA and exhibited very little specificity for origin-containing DNA sequences. It is therefore likely that the active DNA-binding species of T antigen isolated from infected cells is the 5 to 7S form.  相似文献   

12.
Native molecular forms of acetylcholinesterase (AChE) present in a microsomal fraction enriched in SR of rabbit skeletal muscle were characterized by sedimentation analysis in sucrose gradients and by digestion with phospholipases and proteinases. The hydrophobic properties of AChE forms were studied by phase-partition of Triton X-114 and Triton X-100-solubilized enzyme and by comparing their migration in sucrose gradient containing either Triton X-100 or Brij 96. We found that in the microsomal preparation two hydrophilic 13.5 S and 10.5 S forms and an amphiphilic 4.5 S form exist. The 13.5 S is an asymmetric molecule which by incubation with collagenase and trypsin is converted into a 'lytic' 10.5 S form. The hydrophobic 4.5 S form is the predominant one in extracts prepared with Triton X-100. Proteolytic digestion of the membranes with trypsin brought into solution a significant portion of the total activity. Incubation of the membranes with phospholipase C failed to solubilize the enzyme. The sedimentation coefficient of the amphiphilic 4.5 S form remained unchanged after partial reduction, thus confirming its monomeric structure. Conversion of the monomeric amphiphilic form into a monomeric hydrophilic molecule was performed by incubating the 4.5 S AChE with trypsin. This conversion was not produced by phospholipase treatment.  相似文献   

13.
The physical-chemical properties of the nuclear estrogen receptor released by DNase I were characterized. Nuclei were isolated from MCF-7 cells previously exposed to 10-nM-[3H]estradiol. The parameters determined were: sedimentation coefficients (S) on a sucrose gradient, Stokes radii (Rs) by gel filtration on a Sephadex G-200 column and the binding ability to a DNA-cellulose column. The molecular weights (Mr) and frictional ratios (f/fo) were calculated from the S and Rs values. The properties of the receptor released by DNase I obtained from Worthington were compared to the properties of the receptor released by DNase I obtained from Sigma. Digestion with DNase I (Worthington) excised a receptor form which could be solubilized from nuclei by EDTA. This form sedimented at 5.2S with a Rs = 7.08 nm and a calculated Mr = 152.000. About 40% of this receptor form bound to a DNA-cellulose column. 0.4 M KCl dissociated this receptor form into a smaller form sedimenting at 4.2S with Rs = 4.64 nm and a calculated Mr = 80.000. The properties of the receptor solubilized by micrococcal nuclease followed by DNase I (Worthington) digestion were identical to the properties of the DNase I (Worthington) released receptor. Digestion with DNase I (Sigma) released a 3.2S receptor form, which diffused through the nuclear membrane and a 4-5S form which could be extracted from nuclei by EDTA. The 3.2S receptor had a Rs = 2.41 nm, a calculated Mr = 32.000 and less than 5% of it bound to a DNA-cellulose column. Digestion with micrococcal nuclease followed by DNase I (Sigma) solubilized a receptor form with identical properties to the 3.2S receptor. These results suggest that DNase I (Worthington) released a receptor form still associated with some molecules, probably chromatin proteins, which complexed it to DNA, while DNase I (Sigma) released the estradiol binding fragment of the receptor (meroreceptor) as a result of a proteolytic activity present in this preparation.  相似文献   

14.
J M Collins  A K Chu 《Biochemistry》1987,26(18):5600-5607
It is well-known that there are multiple forms of DNA polymerase alpha. In order to determine which form(s) is (are) tightly bound, the activities were dissociated from DNA-poor nuclear matrices, with octyl beta-D-glucoside. Sucrose gradient sedimentation analysis revealed three bands with s values of 7.5, 10.5, and 13. The 7.5S form was free of DNA primase and represented only 10% of the total DNA polymerase alpha bound to the nuclear matrix. The 13S and the 10.5S forms each contained DNA primase activity. The 10.5S form comprised 85% of the DNA polymerase alpha activity and 95% of the DNA primase activity, dissociated from the nuclear matrix. Neither temperature of nuclease digestion nor various salt treatments of nuclei had significant effects on the proportions of DNA polymerase alpha and DNA primase activities bound to, or subsequently dissociated from, nuclear matrices. In a comparison of primase activity bound to the nuclear matrix, dissociated from the nuclear matrix, and in the soluble fraction, it was found that the bound activity had a lower ATP dependence, had less KCl inhibition, and was less sensitive to heat, compared to the dissociated and soluble activities. No differences in Mg2+ or pH dependence were noted. The amounts of DNA polymerase alpha and DNA primase activities bound to the nuclear matrix varied over the cell cycle of synchronized cells. Over the S phase, there were two peaks of matrix-bound DNA primase and two peaks of subsequently dissociated DNA polymerase alpha-DNA primase complex.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

15.
We studied the structure of viral nucleoprotein complexes extracted from the nuclei of mouse cells infected with the immunosuppressive strain of the minute virus of mice (MVMi). Two types of complex were detected, with sedimentation coefficients of about 110 and 40S. The complexes sedimenting at 110S contained single-stranded MVMi DNA as well as a second form of viral DNA which apparently had a heat-sensitive secondary structure. The 110S peak also contained proteins which coelectrophoresed with the MVMi capsid proteins. Complexes sedimenting at 40S contained the double-stranded replicative form of MVMi DNA. These complexes sedimented faster than did the pure replicative form DNA (15S), but more slowly than cellular chromatin fragments containing DNA of the same length. They incorporated labeled deoxynucleoside triphosphate in vitro into the replicative form DNA. We investigated the structure of MVMi nucleoprotein complexes in the following ways. Nuclei of MVMi-infected cells were digested with staphylococcal nuclease, and the resulting DNA fragments were electrophoresed, transferred to nitrocellulose, and hybridized first with labeled MVMi DNA and then with cellular DNA. A nucleosomal repeat pattern was seen with the cellular DNA probe but not with the MVMi DNA probe. The DNA in MVMi nucleoprotein complexes was cross-linked with psoralen, purified, denatured, and examined with an electron microscope. Bubbles, indicating the presence of proteins, were seen in the MVMi DNA. The length of the DNA in the bubbles was 90 +/- 29 nucleotides. On the other hand, nucleosomes protected 160 base pairs from cross-linking by psoralen. The MVMi nucleoprotein complexes thus have a distinct structure which is different from that of chromatin.  相似文献   

16.
To obtain information about the mode of attachment of amphiphilic monomers of acetylcholinesterase (AChE) in sarcoplasmic reticulum (SR) of skeletal muscle, attempts were made to release the enzyme by alkaline hydroxylamine. About half of the activity measured in microsomes preincubated with 0.5% (w/v) Triton X-100 is detached by incubation of SR with bicarbonate buffer (pH 10.5). Addition of 1 M hydroxylamine to the alkaline buffer did not improve enzyme solubilization. Molecular forms of 16S (A12), 10.5S (G4) and 4.0S (G1) are separated by sedimentation analyses of Triton X-100 or bicarbonate-solubilized AChE. Monomeric AChE, released under alkaline conditions (G1A), displays amphiphilic properties. G1A, but not G4 and A12, forms are retained in a phenyl-Sepharose column and this allows its separation from hydrophilic forms. Isolated monomers extracted with Triton X-100 (G1D) or alkaline buffer showed identical kinetic behaviour. The two forms reacted with lectins in a similar manner. However, thermal inactivation experiments revealed that about 90 and 40% of the activity in the G1D and G1A forms were lost by heating at 50 degrees C, following the same rate constant (k = 0.130 min-1). Addition of Triton X-100 to the G1A form leads to an increase of its thermal sensitivity, the enzyme being fully inactivated very rapidly (k = 0.230 min-1). The results suggest that the hydrophobic moiety of the enzyme might be exposed or hidden depending on the environmental hydrophobicity. Changes in the composition of the solvent will determine the final conformational state of the protein.  相似文献   

17.
The biosynthesis and secretion of dopamine beta-hydroxylase were investigated by radiolabeling rat pheochromocytoma (PC12) cells in culture. Intracellular dopamine beta-hydroxylase from a crude chromaffin vesicle fraction and secreted dopamine beta-hydroxylase from culture medium were immunoprecipitated using antiserum made against purified bovine soluble dopamine beta-hydroxylase. Analysis of the immunoprecipitated enzyme on sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that: 1) the membrane-bound form of the hydroxylase from crude secretory vesicle membrane extracts contained two nonidentical subunits in approximately stoichiometric amounts (Mr = 77,000 and 73,000); 2) the soluble hydroxylase from the lysate of these secretory vesicles was composed predominantly of a single subunit (Mr = 73,000); and 3) the hydroxylase secreted into the medium under resting conditions was also composed of a single subunit (approximate Mr = 73,000). All subunits of the multiple forms of hydroxylase were glycoproteins. Under resting conditions, the rate of secretion of hydroxylase was approximately 6% of total cellular enzyme/15 min. The secreted form of the hydroxylase incorporated [35S]sulfate, whereas no significant [35S]sulfate was incorporated into the cellular forms of enzyme. We propose that in addition to the dopamine beta-hydroxylase which is found in catecholamine storage vesicles and released during stimulus-coupled exocytosis, PC12 cells also have a constitutive secretory pathway for dopamine beta-hydroxylase and that the enzyme released by this second pathway is sulfated.  相似文献   

18.
Abstract: Rat brain was found to enzymatically methylate phospholipids to form phosphatidylcholine with S -adenosyl- l -methionine serving as the methyl donor. Methyltransferase activity was localized in the microsomes and synaptosomes. In synaptosomes, at least two enzymes were found to be involved in the formation of phosphatidylcholine. The first methyltransferase which catalyzes the methylation of phosphatidylethanolamine to form phosphatidyl- N -monomethylethanolamine was found to have a pH optimum of 7.5, a low Km for 5-adenosyl- l -methionine and a partial requirement for Mg2. Methyltransferase I is tightly bound to membranes. The second methyltransferase (II) catalyzes the successive methylations of phosphatidyl- N -monomethylethanolamine to phosphatidyl- N , N -dimethylethanolamine and then to phosphatidylcholine. In contrast to methyltransferase I, methyltransferase II has a pH optimum of 10.5, a high apparent Km for S -adenosyl- l -methionine and no requirement for Mg2. Methyltransferase II is easily solubilized by sonication. The highest specific activity for both enzymes was found in the synaptosomal plasma membrane.  相似文献   

19.
Human leiomyoma of the uterus contained seven forms of cyclic AMP phosphodiesterase in the crude cytosol as revealed by a specific activity stain on non-denaturing polyacrylamide gel electrophoresis. The enzymes from human myometrium and normal uterus showed an identical activity pattern. Ferguson plot analysis showed four different molecular weight species of Mr 229,000 +/- 4,000, 186,000 +/- 4,000, 174,000 +/- 4,000 and 162,000 +/- 4,000. The Mr 174,000 species comprised four differently charged forms. Sucrose density gradient centrifugation of the crude cytosol revealed the presence of three molecular weight species sedimenting at 11.8S, 8.1S and 3.6S. The Michaelis constant (Km) for the band 1 form which displayed linear kinetics was 5 microM and the band 2 form which produced non-linear kinetics had Km values of 5.8 and 37 microM.  相似文献   

20.
The mouse neuroblastoma cell line NB2A produces cellular and secreted acetylcholinesterase (AChE). After incubation of the cells for 4 days the ratio between AChE secreted into the medium and AChE in the cells was 1:1. The cell-associated enzyme could be subdivided into soluble AChE (25%) and detergent-soluble AChE (75%). Both extracts contained predominantly monomeric AChE (4.6S) and minor amounts of tetrameric AChE (10.6S), whereas the secreted AChE in the culture supernatant contained only the tetrameric form. All forms were partially purified by affinity chromatography. It could be demonstrated that the secretory and the intracellular soluble tetramers were hydrophilic, whereas the detergent-soluble tetramer was an amphiphilic protein. On the other hand the soluble and the detergent-soluble monomeric forms were amphiphilic and their activity depended on the presence of detergent. By digestion with proteinase K amphiphilic monomeric and tetrameric AChE could be converted to a hydrophilic form that no longer required detergent for catalytic activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [3H]diisopropylfluorophosphate-labelled AChE gave one band at 64 kilodaltons (kD) under reducing conditions and two additional bands at 120 kD and 140 kD under nonreducing conditions.  相似文献   

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