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Following the in vitro studies described above, we analysed the in vivo binding characteristics of NxMBD proteins. The proteins were expressed in mouse fibroblasts and detected using an anti-HA antibody. As expected, the wild-type proteins co-localized with methyl-CpG-dense heterochromatic foci . 4xMBD showed significantly more intense staining than 1xMBD, in agreement with the in vitro analysis. The negative control proteins (R22A) did not co-localize with the heterochomatic foci (data not shown). As a further control for the specificity of in vivo binding, we found that heterochromatic localization of wild-type 4xMBD proteins was lost in cells that lack Dnmt1 ( 17 ) and, hence, have greatly reduced levels of CpG methylation .
|
16893950_p15
|
16893950
|
In vivo binding of multimeric DNA to methyl-CpG sites
| 4.107165 |
biomedical
|
Study
|
[
0.9994465708732605,
0.0002816584019456059,
0.0002718073083087802
] |
[
0.9995324611663818,
0.00016074681479949504,
0.0002462842676322907,
0.00006050542651792057
] |
en
| 0.999997 |
To determine whether 4xMBD could recruit a functional domain to non-heterochromatic methylated sites in vivo , the 4xMBD protein was fused to the VP16-activation domain and expressed in cells that also received reporter constructs. As shown in Figure 5C , the 4xMBD–VP16 fusion caused a ∼100-fold increase in expression from a methylated reporter, whereas unfused 4xMBD protein had no effect. A slight induction (∼2-fold) of the non-methylated construct by 4xMBD–VP16 was observed, which may be due to non-specific effects of its strong activation domain. Staining of cells transfected with the 4xMBD–VP16 construct demonstrated that the overall localization of the fusion protein is indistinguishable from that of non-fusion 4xMBD . We conclude that MBD is available to interact with euchromatic methylated sites and can recruit a functional domain to these sites in vivo .
|
16893950_p16
|
16893950
|
In vivo binding of multimeric DNA to methyl-CpG sites
| 4.224058 |
biomedical
|
Study
|
[
0.999452531337738,
0.0002821359084919095,
0.0002653428527992219
] |
[
0.9993619322776794,
0.00025578707573004067,
0.0003132417914457619,
0.00006906418275320902
] |
en
| 0.999997 |
We demonstrate here that the 4xMBD polypeptide has a high affinity for methyl-CpG sites in vitro and in vivo . Multimerization significantly amplifies the affinity without loss of specificity as measured by interaction with (i) methylated DNA in solution, (ii) DNA immobilized on membranes and (iii) chromosomal DNA in cytological preparations. These methods consistently suggest a ∼100-fold enhancement of binding due to tetramerization compared with the monomeric MBD. In spite of this improved binding, we saw little evidence for loss of specificity, as non-methylated DNA was negative for interaction with the 4xMBD polypeptide by all assays. Available antibodies raised against the m 5 C base require DNA to be denatured by heat or alkali in order to fully expose the epitope. An advantage of the poly-MBD proteins is that they recognize methyl-CpG in duplex DNA and therefore do not require cytological preparations to be subjected to harsh denaturing conditions that may compromise specimen integrity. Also, 4xMBD can be produced in the laboratory simply and relatively cheaply by expression in bacteria. Poly-MBD proteins can therefore be considered as an alternative to anti-m 5 C antibodies as sensitive and specific reagents for the detection of methyl-CpG sites in genomic DNA.
|
16893950_p17
|
16893950
|
CONCLUSIONS
| 4.477273 |
biomedical
|
Study
|
[
0.9993558526039124,
0.0004303230671212077,
0.0002137999836122617
] |
[
0.9975959658622742,
0.0005441322573460639,
0.001695128041319549,
0.00016477498866152018
] |
en
| 0.999997 |
The CACCC box was initially recognized as a regulatory element by sequence homology analysis and mutation assays ( 1 – 7 ) and is found in a large number of genes, in a broad spectrum of species, including plants ( 8 ). The CACCC box usually resides 100–200 bp 5′ to the TATA box. Disruption of the CACCC box results in diminution of promoter activity in in vitro transcription or in cell transfection assays, suggesting that the CACCC box positively contributes to the promoter strength. Recently, it was reported that several CACCC-binding factors could function as repressors ( 9 ). However, a mutation that increases gene expression by disrupting the CACCC box has not been found either using a reporter gene assay or in vivo .
|
16914453_p0
|
16914453
|
INTRODUCTION
| 4.251524 |
biomedical
|
Study
|
[
0.9994627833366394,
0.00020496516663115472,
0.00033225343213416636
] |
[
0.9962198138237,
0.00030133393011055887,
0.003401600755751133,
0.00007721879228483886
] |
en
| 0.999995 |
To date, the in vivo function of the CACCC box has been assessed only in the globin gene locus. A CACCC box is present in all the promoters of the globin genes and in DNase I hypersensitive sites (HS) 2, 3 and 4 of the β locus control region (LCR) ( 10 ). The functional significance of the β gene CACCC box has been well established by various assays, and by in vivo evidence that mutations of this regulatory element result in the phenotype of β thalassemia ( 11 ). A β-globin gene activator, EKLF, has been identified to bind specifically to the CACCC box of the β-globin gene ( 12 ). In the mouse, homozygosity for EKLF gene deletions is lethal by days 14–15 of fetal development due to the absence of β-globin gene expression and effects on other genes of the definitive erythropoiesis ( 13 , 14 ). Thus, these results unequivocally validated the hypothesis that the CACCC box functions as a positive promoter element in vivo . The function of the γ-globin gene CACCC-box has been studied in the context of the competition model of hemoglobin switching, which hypothesizes that the expression of the γ-globin gene precludes expression of the β gene in embryonic erythropoiesis by competing for the enhancing activity of the LCR. Townes and co-workers ( 15 ) demonstrated that an intact human γ-globin gene CACCC box was essential for correct human β-globin gene regulation in transgenic mice. When this site is mutated in the context of a cosmid construct containing the 22 kb LCR, the β-globin gene is expressed at high levels in embryonic erythroid cells. In addition, this study showed that disruption of the γ CACCC box did not abolish γ-gene expression in the embryos. Similar results were obtained in the transgenic mice carrying a HS2/HS3γβ plasmid construct ( 16 ). In transgenic mice carrying the plasmid construct, the γ gene was highly expressed in definitive erythroid cells. Disruption of the CACCC box abolished γ-gene expression in the fetal and adult stages of development, whereas it reduced γ-gene expression by ∼3-fold in the 10.5 day yolk sac.
|
16914453_p1
|
16914453
|
INTRODUCTION
| 4.60996 |
biomedical
|
Study
|
[
0.9991301894187927,
0.0005016494542360306,
0.0003681608650367707
] |
[
0.9958686828613281,
0.0003775327932089567,
0.0035609707701951265,
0.00019283407891634852
] |
en
| 0.999997 |
To test the role of the CACCC box in the transcriptional potential of the γ-globin gene promoter in vivo , we produced deletions and mutations of the γ-gene promoter and examined their effects in transgenic mice. Our findings show that the CACCC box is not required for γ-gene activation in primitive erythropoiesis but it is necessary for γ-gene expression in the cells of definitive erythropoiesis. The CACCC mutations diminished DNase I hypersensitivity and the recruitment of polII and TBP at the γ-gene promoter in adult erythropoiesis; however, they had only slight or no effect on the acetylation status of histone H3 and H4. These results are similar to our previous findings showing that the γ-gene expression in embryonic erythroid cells is not disrupted by the CCAAT or TATA box mutations ( 17 , 18 ). Taking these findings together, we hypothesize that the trans factors recruited by the CACCC, CCAAT and TATA boxes interact with each other to form a large complex, which in the following discussion we designate as the ‘promoter complex’. It is likely that in the embryonic stage of development, functions of a trans factor which fails to be recruited into the complex because of a mutated promoter box is compensated by those factors recruited by the remaining cis elements. As a results, the LCR interacts with this incomplete ‘promoter complex’ in embryonic erythroid cells. In contrast, an intact ‘promoter complex’ is required for the interaction with the LCR in adult erythroid cells.
|
16914453_p2
|
16914453
|
INTRODUCTION
| 4.364889 |
biomedical
|
Study
|
[
0.9994004964828491,
0.00040705964784137905,
0.0001923527306644246
] |
[
0.998958945274353,
0.00033175863791257143,
0.0005912418710067868,
0.00011801477376138791
] |
en
| 0.999998 |
The plasmid constructs, μLCR–382 A γ(ΔCAC −138 to −156) (construct A), μLCR–382 A γ(−142 CC→TG) (construct B) and μLCR–382 A γ(−140 A→C) (construct C) were constructed by standard oligonucleotide-directed mutagenesis (Promega) on the basis of the parental construct μLCR–382 A γ. ( 19 ). The mutations were verified by DNA sequencing. The construct DNA fragment was relieved from the plasmid backbone by restriction enzyme digestion and separated by electrophoresis on 0.8% agarose gel. The fragment was recovered from the gel by freeze–thaw cycle and filtered from an Ultrafree MC tube (Millipore). The DNA fragment was further purified by phenol and chloroform extraction and precipitated with ethanol. The DNA was resuspended in the low TE buffer (10 mM Tris–HCl, pH 7.5 and 0.1 mM EDTA) and diluted to 1–3 ng/μl. The DNA solution was filtered again through a syringe filter (Corning) just before microinjection.
|
16914453_p3
|
16914453
|
Constructs
| 4.169323 |
biomedical
|
Study
|
[
0.9995200634002686,
0.0002589616924524307,
0.00022101719514466822
] |
[
0.9987788796424866,
0.0008535197703167796,
0.00028053976711817086,
0.0000870760777615942
] |
en
| 0.999998 |
Purified DNA fragments were injected into fertilized mouse eggs (B6/C3F1) and then transferred to pseudopregnant foster mother (B6/D2F1). Founder animals were identified by slot blotting with a HS3 probe. F1 progeny were obtained by breeding founder animals with non-transgenic mice (B6/D2F1) and were screened for correct integration and to exclude the presence of mosaicism in the founders. To study the developmental pattern of human γ-gene expression, staged pregnancies were interrupted on day 12 of development and samples from blood yolk sac and fetal liver were collected.
|
16914453_p4
|
16914453
|
Transgenic mice
| 4.106166 |
biomedical
|
Study
|
[
0.9995294809341431,
0.00025782737066037953,
0.0002127371117239818
] |
[
0.9992309808731079,
0.0004387937078718096,
0.0002583077293820679,
0.00007189499592641369
] |
en
| 0.999996 |
DNA from fetal brain or carcass of F2 progeny in each line was isolated by standard procedures. At least three DNA samples were obtained from each line. Individual samples were then digested with a restriction enzyme. DNA (10 μg) from each enzyme reaction performed on samples from a given line was loaded onto a 0.8% agarose gel, and DNA fragments were resolved by electrophoresis. Southern blot hybridization was performed with a BamHI–EcoRI probe derived from the large intron of the γ-globin gene. Signals were quantified on a PhosphorImager. The blot was then striped and rehybridized with a mouse α-globin gene probe and the intensity of the signals on each lane was used for DNA loading correction. Copy numbers were determined by comparing the signal from a given transgenic line with those of human genomic DNA. In cases in which the computed value was not an integer, the copy number was rounded to the nearest integer in standard fashion.
|
16914453_p5
|
16914453
|
DNA analysis
| 4.148843 |
biomedical
|
Study
|
[
0.9995406866073608,
0.0002673366107046604,
0.00019197868823539466
] |
[
0.9990792274475098,
0.00047758041182532907,
0.0003681484377011657,
0.00007500696665374562
] |
en
| 0.999999 |
Total RNA was prepared from the tissues containing the primitive erythrocytes (d12 blood and yolk sac) and the tissues containing the definitive erythrocytes (d12 fetal liver and adult blood). RNA samples were separately isolated from three or more transgenic individuals from each time point. The human γ-globin and murine α- and ζ-globin mRNA was detected by RNase protection assay and quantified by a PhosphorImager. To minimize experimental error, samples from individual animals were quantified independently and multiple measurements were performed in RNase protection. Copy number-corrected globin mRNA levels were expressed as human γ mRNA/γ copy number/(murine ζ mRNA/2 + murine α mRNA/4).
|
16914453_p6
|
16914453
|
RNA analysis
| 4.115264 |
biomedical
|
Study
|
[
0.9995821118354797,
0.00021864048903807998,
0.00019920185150112957
] |
[
0.999308705329895,
0.0003002281882800162,
0.00033411808544769883,
0.000056873490393627435
] |
en
| 0.999997 |
Single-cell spleen suspensions were prepared from phenylhydrazine-treated 10- to 12-week-old transgenic mice. Nuclei isolation, DNase I digestion and Southern blotting were performed as described previously ( 20 ). The probe shown in Figure 1 was a BamHI/EcoRI fragment encompassing intron 2 of the γ gene. DNase I was purchased from Worthington Biochemical Corporation (Lakewood, NJ).
|
16914453_p7
|
16914453
|
DNase I hypersensitive assay
| 4.049558 |
biomedical
|
Study
|
[
0.9996216297149658,
0.00015525407798122615,
0.00022306072060018778
] |
[
0.9988334774971008,
0.0008346723625436425,
0.00026709423400461674,
0.00006481978198280558
] |
en
| 0.999997 |
Single-cell spleen suspensions were prepared from 10- to 12-week-old transgenic mice after 4 days of phenylhydrazine-induced hemolytic anemia. ChIP assays were performed as described previously with modifications ( 17 ). The mousethe β maj globin gene was selected as internal controls. Immunoprecipitations (IP) were performed at least three times on different days. Each DNA was diluted at three dilutions and the PCR readings of the three dilutions should be within the range of the standard DNA curve. PCR was performed on an Opticon 2 (MJ Research). All data were expression as ratio of the PCR readings of a given primer set over the internal control and SD was shown. Rabbit polyclonal antibodies against histone H3 acetylated at lysines 9 and 14 and H4 at lysines 5, 8, 12 and 16 were purchased from Upstate Biotechnology (Lake Placid, NY). Rabbit polyclonal antibodies against Pol II (N-20, sc-899) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Syber green PCR kit was from Qiagen (Valencia, CA).
|
16914453_p8
|
16914453
|
Chromatin immunoprecipitation assay (ChIP)
| 4.077487 |
biomedical
|
Study
|
[
0.999652624130249,
0.0001644060539547354,
0.00018295887275598943
] |
[
0.9991750121116638,
0.0004502476949710399,
0.0003121015033684671,
0.00006270292215049267
] |
en
| 0.999997 |
MEL nuclear extracts were prepared as described by Andrews and Faller ( 21 ). In detail, ∼3 × 10 7 logarithmic-phase MEL cells were harvested and washed once with ice-cold PBS. The cells were then incubated in 2 ml of ice-cold Buffer A [10 mM HEPES–KOH, pH 7.9, 1.5 mM MgCl 2 , 10 mM KCl, 0.5 mM DTT and 0.2 mM phenylmethylsulfonyl fluoride (PMSF)] for 10 min on ice. The suspension was centrifuged at 10 000 g for 10 s. The pellet was resuspended in 500 μl ice-cold buffer C (20 mM HEPES–KOH, pH 7.9, 25% glycerol, 1.5 mM MgCl 2 , 420 mM NaCl, 0.2 mM EDTA, 0.5 mM DTT and 0.2 mM PMSF) and incubated on ice for 30 min. The crude nuclear extract was obtained after the cellular debris was removed by centrifugation at 12 000 g for 30 min at 4°C. Protein concentration was determined by using the Bio-Rad protein assay kit. The labeled probes (1 × 10 4 c.p.m.) were incubated with 5 μg MEL nuclear extracts for 20 min at room temperature, in the binding buffer containing 10 mM Tris–HCl, pH 7.5, 50 mM NaCl, 0.5 mM DTT, 10% glycerol, 1 μg poly(dI–dC), 0.05% NP-40 and competitors of various-concentration. Samples were electrophoresed in 4.5% polyacrylamide gel in 0.5× Tris–borate–EDTA (TBE) buffer containing 4 mM Mg 2+ at 4°C. The oligonucleotide sequences used in the assays was shown in Figure 4A.
|
16914453_p9
|
16914453
|
Gel shift assay
| 4.148329 |
biomedical
|
Study
|
[
0.9995009899139404,
0.00025827137869782746,
0.00024066766491159797
] |
[
0.9993095397949219,
0.0003230791771784425,
0.0003107578959316015,
0.00005662280454998836
] |
en
| 0.999996 |
Truncation of the γ-gene promoter to position −141 γ abolishes γ-gene expression in the adult red cells of transgenic mice, presumably because this truncation destroys the γ-gene CACCC box ( 19 ). Extension of the γ-gene promoter to −159 γ (which includes the γ CACCC box) results in a 28-fold increase in γ mRNA production compared to the −141 A γ transgenic mice (γ/α mRNA increased from 0.5 ± 0.3 to 14.3 ± 3.3) ( 19 ). To further test the role of the γ CACCC box on γ-gene expression, we deleted an 18 bp sequence (from −138γ to −156γ) encompassing theCACCC box in the context of the −382γ promoter. Five transgenic lines ( Table 1 ) carrying the μLCR(−382) A γ(ΔCAC) construct were established and γ-gene expression was measured during development. As shown in Table 1 (construct A), deletion of the CACCC box region results in reduction of γ-gene expression in the fetal and adult stages of definitive erythropoiesis. γ mRNA levels in blood of adult μLCR(–382) A γ(ΔCAC) mice were 16-fold lower than in the μLCR(−382) A γ control mice. These results provide in vivo evidence that the CACCC box region is required for γ-gene expression in the definitive erythropoiesis of fetal liver and it is necessary for γ-gene expression in adult red cells.
|
16914453_p10
|
16914453
|
The CACCC box is required for human γ-globin gene expression in the adult but not in the embryonic erythropoiesis
| 4.289675 |
biomedical
|
Study
|
[
0.9994496703147888,
0.0003431238583289087,
0.00020717945881187916
] |
[
0.9992713332176208,
0.0003155658196192235,
0.00031051674159243703,
0.00010250424384139478
] |
en
| 0.999998 |
The deletion of the 18 bp CACCC region removed more base pairs than required for a binding site of a putative Krűppel-like transcription factor, which recognizes a 9 bp motif. This raised the possibility that the phenotype observed in the CACCC deletion mice might be due to the removal of other DNA-binding sites in the region that are essential for γ expression. To address this question we destroyed the γ CACCC binding activity by introducing point mutations into the box (CACCC→CA TG C). These mutations destroyed the protein-binding activity of the CACCC box as determined by gel shift assay (see below). The mutation did not impair γ-gene expression in embryonic erythropoiesis (blood and yolk sac at d12) in the five transgenic lines mice carrying this construct ( Table 1 , construct B). In contrast, the mutation resulted in striking decrease of γ-gene expression in adult erythropoiesis. Thus, γ mRNA in the adult blood of the CACCC mutant mice was reduced to 1.1% of murine α mRNA, i.e. to the same level as that in the mice carrying the deletion of the CACCC region. These results confirm that the γ CACCC box per se is essential for γ-gene expression in adult erythropoiesis.
|
16914453_p11
|
16914453
|
The CACCC box is required for human γ-globin gene expression in the adult but not in the embryonic erythropoiesis
| 4.355157 |
biomedical
|
Study
|
[
0.9994352459907532,
0.0003452109813224524,
0.0002195230044890195
] |
[
0.9992161989212036,
0.00037132727447897196,
0.0002896855876315385,
0.00012278104259166867
] |
en
| 0.999996 |
In contrast to its role in γ-gene expression in definitive cells, the CACCC box region is not required for γ-gene expression in embryonic cells. As shown in Table 1 , there is essentially no difference in γ mRNA levels between the wild-type control and the CACCC deleted or mutant transgenic mice in embryonic blood and yolk sac samples.These results show that the γ gene can be efficiently expressed in embryonic cells in the absence of a γ CACCC box. The non-essential role of the CACCC box in γ-gene expression at the embryonic stage of development has been reported by Ryan et al . ( 15 ) and Sargent and Lloyd ( 16 ).
|
16914453_p12
|
16914453
|
The CACCC box is required for human γ-globin gene expression in the adult but not in the embryonic erythropoiesis
| 4.135702 |
biomedical
|
Study
|
[
0.9994872808456421,
0.00020197322010062635,
0.00031079008476808667
] |
[
0.9994044303894043,
0.00025448037195019424,
0.00028902399935759604,
0.00005200291707296856
] |
en
| 0.999996 |
To delineate the mechanistic relationship between the CACCC box and γ gene activation, we compared the chromatin conformation in transgenic mice carrying the wild type and in mice carrying the −142 CC→TG CACCC box mutant constructs. The DNase I hypersensitivity assay was performed on spleens of phenylhydrazine-treated adult transgenic mice. This treatment induces acute anemia, resulting in a great expansion of erythropoiesis in the spleen so that erythroblasts account for >80% of splenic cells. Figure 1 shows Southern blot hybridization results of DNase I sensitivity assay. In wild-type mice, HS sites were formed in the LCR as well as in the γ gene promoter . The CACCC mutation disrupted HS formation at the γ-gene promoter, but had no effect on the LCR region . These results suggest that the effect of the CACCC mutation on chromatin structure is confined to the γ-gene region.
|
16914453_p13
|
16914453
|
Effects of the γ CACCC mutant on chromatin structure
| 4.186069 |
biomedical
|
Study
|
[
0.9995008707046509,
0.0003061390307266265,
0.00019299774430692196
] |
[
0.9993948936462402,
0.00024474819656461477,
0.0002821118396241218,
0.00007822592306183651
] |
en
| 0.999996 |
Effects of the γ CACCC mutation on histone acetylation were determined by ChIP assays using antibodies against histone H3 (acetylated K9, 14) or H4 (acetylatedK5, 8, 12, 16). Levels of histone acetylation were expressed as fold over that at the β maj gene promoter, i.e. an endogenous murine gene in which the histone acetylation level should be same in the wild type and in the CACCC mutant transgenic mice. As shown in Figure 2A the CACCC mutation did not affect the acetylation levels of histone H3 in the LCR region, whereas it slightly reduced acetylation levels in the γ-gene region. The differences in the promoter, exon 2, and 3′ region between the wild type and the CACCC mutant are statistically significant ( P < 0.05, from eight independent ChIP experiments). On the other hand, the CACCC mutation did not have any effects on histone H4 acetylation either in the LCR or in the γ-gene region .
|
16914453_p14
|
16914453
|
Effects of the γ CACCC mutant on chromatin structure
| 4.143508 |
biomedical
|
Study
|
[
0.9995132684707642,
0.00024517811834812164,
0.0002414989867247641
] |
[
0.9994920492172241,
0.00015661554061807692,
0.00029318153974600136,
0.000058033027016790584
] |
en
| 0.999998 |
Taken together, these results suggest that the effects of the CACCC mutation on chromatin conformation are restrained to the γ-gene region.
|
16914453_p15
|
16914453
|
Effects of the γ CACCC mutant on chromatin structure
| 3.675775 |
biomedical
|
Study
|
[
0.9989925026893616,
0.0002089357003569603,
0.0007986488053575158
] |
[
0.9962922930717468,
0.003130710683763027,
0.0004537026397883892,
0.00012325348507147282
] |
en
| 0.999998 |
The CACCC mutation severely impaired recruitment of pol II and TBP at the γ gene promoter. As shown in Figure 2C and D , pol II binding at the γ-gene promoter in the control mice was equivalent to 21 ± 0.2% of that at the mouse β maj gene promoter, whereas the corresponding measurement in the CACCC mutant mice was 3.4 ± 0.8% of that at the β maj gene promoter. Thus, the CACCC mutation resulted in an 86% reduction of pol II recruitment at the γ-gene promoter. Similarly, the CACCC mutation caused an 88% reduction of TBP binding at the γ-gene promoter in comparison with the wild-type control.
|
16914453_p16
|
16914453
|
Effects of the CACCC mutation on pol II and TBP recruitment
| 4.189137 |
biomedical
|
Study
|
[
0.9995379447937012,
0.00024431070778518915,
0.00021786343131680042
] |
[
0.9993504881858826,
0.000317184574669227,
0.00025858855224214494,
0.00007368493970716372
] |
en
| 0.999997 |
The effects on the CACCC mutant on recruitment of pol II and TBP extended to the LCR region. As shown in Figure 2C and D , the TBP recruitment on HS1-4 cores of the LCR region was reduced, on average, by the CACCC box mutation to 15% of the wild-type control; the pol II recruitment was reduced to 28% of the wild type.
|
16914453_p17
|
16914453
|
Effects of the CACCC mutation on pol II and TBP recruitment
| 4.110825 |
biomedical
|
Study
|
[
0.9993482232093811,
0.0002481296833138913,
0.00040366844041273
] |
[
0.9994146823883057,
0.00034984375815838575,
0.00017995793314184994,
0.00005561784200835973
] |
en
| 0.999995 |
In a previous study, we demonstrated that replacement of the human γ CACCC box with either the human ɛ or the galago γ CACCC box resulted in a downregulation of γ-gene expression in adult erythropoiesis, suggesting that the γ CACCC box contributes both to the activation and to the silencing of the γ gene in the adult stage of development ( 22 ). It is presumed that the trans factors that bind to the CACCC box of the globin genes are putative Krűppel-like transcription factors with three zinc-finger motifs, such as EKLF. The zinc-finger motifs recognize a 9 bp sequence, CCN CNC CCN. In the human γ-gene promoter, this particular sequence is CTC CAC CCA. We noticed that the common base in the ‘embryonic’ CACCC box of the human ɛ and galago γ promoters is C at the ninth position, whereas it is A in the human γ CACCC box . To test whether this difference could be responsible for the developmental specificity of the CACCC boxes, a A→C transversion at the −140 position was introduced into the γ CACCC box in the context of the μLCR(−382) A γ construct and transgenic mice were produced. Seven lines were established. Quantitative data of γ-gene expression in embryonic and definitive erythrocytes are presented in Table 1 (construct C). The mean level of γ-gene expression in yolk sac of the seven lines was 25.8 ± 7.6%. The corresponding data in the wild-type control was 21.0 ± 7.4%; the difference was not statistically significant ( P > 0.5). In adult blood, the γ gene was expressed at 12.8 ± 5.0% in the −140 mutant mice, a value similar to that of the wild-type control. We conclude that the −140 A→C change has no effect on γ-gene expression. However, we noticed that the distribution of γ-gene expression in the −140 A→C mutant transgenic lines was much diverged. Figure 3A and B show the distribution of γ-gene expression in individual transgenic mice carrying the −140 A→C mutant along with corresponding data of the CACCC inactivation mutant (−142 CC→TG) and the wild-type control. In embryonic erythropoiesis , the distribution of γ-gene expression in the −140 A→C mutant mice was similar to those of the wild type and the CACCC inactivation mutant transgenic mice, except that it was slightly wider compared to other two. In adult erythropoiesis, the levels of γ-gene expression could be separated into two groups . In five lines, the mean level of γ-gene expression was similar to wild-type control while two other lines expressed the γ gene at a level similar to that of the CACCC box inactivated mice and of replacements of the human ɛ or the galago γ CACCC box ( 22 ). These results suggest that the 1 bp mutation in the γ CACCC box results in an increase in susceptibility of the gene to position effects from the surrounding chromatin.
|
16914453_p18
|
16914453
|
Search for the sequences responsible for developmental specificity in the γ CACCC box
| 4.331274 |
biomedical
|
Study
|
[
0.9993347525596619,
0.00039144381298683584,
0.000273735320661217
] |
[
0.9993638396263123,
0.00021513196406885982,
0.0003320666146464646,
0.00008907303708838299
] |
en
| 0.999998 |
It is likely that the functional specificities of the various CACCC boxes reflect their distinct binding activity. Figure 4A shows six different CACCC boxes that have been tested in transgenic mice. The 9 bases that comprise the binding motif of a putative zinc-finger protein are in capital letters and numbered from 1 to 9 on top. Mutated base pairs that were introduced into the wild-type human γ CACCC sequence are boxed. Nucleotide differences between the human γ gene and the human ɛ gene or the galago γ gene CACCC boxes are shown in italics. In the context of the μLCR γ constructs, the presence of the human γ CACCC box was associated with an HPFH-like phenotype, i.e. the γ gene is highly expressed during adult erythropoiesis. The CC→TG mutation of the CACCC box abolished γ-gene expression in adult transgenic mice. Introduction of the ‘down’ mutation (A→C at position 9) resulted in a high susceptibility of the gene to its surrounding chromatin environment, while an ‘up’ T→C transition at position −4 has no effect on γ-gene expression (data not shown). Replacement of the human γ CACCC box with the human ɛ gene or galago γ gene CACCC box resulted in an ‘embryonic’ pattern of expression ( 22 ). Figure 4B shows the binding patterns of the six CACCC box oligonucleotides in get shift assays using MEL nuclear extract. Except for the CC→TG mutation, which abolished binding activity, all oligonucleotides had a similar binding pattern, suggesting that they were able to bind identical or very similar factors. However, the intensity of the major shifted band could be distinguished between different oligonucleotides, suggesting that the binding affinities of these sequences were different. The ‘up’ T→C mutation, which did not result in a phenotype change, had similar binding affinity as the wild-type γ CACCC box. On other hand, the ‘down’ A→C mutation, which resulted in an increase of position effect of γ-gene expression, and the human ɛ gene or the galago γ gene CACCC box, which resulted in an ‘embryonic’ expression pattern, had a much higher binding activity in comparison with the wild-type γ CACCC box. To confirm these observations, quantitative competition assays were performed. 32 P-labeled oligonucleotides of the wild type, down mutation, galago γ and human ɛ CACCC boxes in the same fixed chemical amount were competed with different amounts of the cold self competitors in gel shift assays . The retarded bands were quantified by PhosphorImager and the resulting data were plotted against the amount of the competitors . The results demonstrated that the wild-type γ CACCC box has a lower binding affinity compared to the ‘down’ mutation, the human ɛ and the galago γ CACCC boxes.
|
16914453_p19
|
16914453
|
Binding activity of the CACCC boxes
| 4.34556 |
biomedical
|
Study
|
[
0.9992904663085938,
0.0003854505775962025,
0.0003239801153540611
] |
[
0.9992691874504089,
0.0002207654033554718,
0.0004221707349643111,
0.0000878989594639279
] |
en
| 0.999998 |
Developmentally proper regulation of the globin genes depends on at least two factors: first, the developmental specificity of the globin gene promoters; second, the spatial relationships between the LCR and genes. Alterations in the developmental profiles of globin gene expression have been observed in many transgenic studies using artificial constructs, including YACs, BACs and cosmids. These changes provide great opportunities for mechanistic studies of hemoglobin switching. However, to study a complex phenomenon like hemoglobin switching, the ideal approach is a reductionist approach dissecting the event into several parameters and analyzing them one by one. This approach was applied in the transgenic model we used in these studies. In LCR γ gene mice, the enhancing activity of the LCR is continually available for the γ gene, most likely because of the short distance between the enhancer and the gene, which spares the requirement of loop formation. Therefore, the system allows us to study the effect of promoter mutations on the transcriptional potential of the γ gene, while the parameter of enhancer activity/loop formation remains constant. Using this system we obtained evidence that the CACCC box is necessary for the activation of the γ gene in definitive erythropoiesis, but is not required for the activation of γ-gene expression in primitive cells. Similar results have been reported by Townes ( 15 ) and Sargent and Lloyd ( 16 ).
|
16914453_p20
|
16914453
|
DISCUSSION
| 4.369431 |
biomedical
|
Study
|
[
0.9995371103286743,
0.00027265987591817975,
0.00019032567797694355
] |
[
0.9985948204994202,
0.00030516344122588634,
0.0010029698023572564,
0.00009702952957013622
] |
en
| 0.999997 |
The CACCC box-independent activation of the γ-globin gene in embryonic erythroid cells is an unexpected finding in view of the fact that this motif has been documented as a positive cis element in all tested CACCC box-containing promoters. Our findings suggest that the trans factor milieu in embryonic erythroid cells can support the activation of the CACCC box-less γ gene, and the LCR is able to enhance transcription from the CACCC box-less γ-gene promoter. We have demonstrated previously that disruption of the TATA box has no effect on γ-gene activation in embryonic erythropoiesis ( 17 ). Furthermore, the CCAAT box mutation only moderately impairs γ-gene expression in the embryonic stage ( 18 ). We hypothesize that the trans factors bound at the γ-gene promoter interact with each other to form a ‘promoter complex’, which functions as a single entity to interact with the LCR. In embryonic erythroid cells the LCR is able to interact with the ‘promoter complex’ even if a component normally binding with one of the promoter motifs is missing. In contrast, in adult erythropoiesis the LCR can only interact with a complete ‘promoter complex’.
|
16914453_p21
|
16914453
|
DISCUSSION
| 4.448103 |
biomedical
|
Study
|
[
0.9993932247161865,
0.00036183124757371843,
0.00024489255156368017
] |
[
0.9990096092224121,
0.00038054102333262563,
0.0004984711413271725,
0.00011125715536763892
] |
en
| 0.999996 |
Consistent with the abolishment of γ-gene expression, the CACCC mutation resulted in the diminution of HS formation and TBP and polII recruitments at the γ-gene promoter in adult erythroid cells. In contrast, the γ CACCC mutation resulted in only a slight or no effects on histone H3 and H4 acetylation, suggesting that the activation of the γ-globin gene is not directly related to the level of histone acetylation. On the other hand, a positive relationship between histone acetylation and globin gene activation has been established in the context of the entire globin locus ( 23 – 25 ). This discrepancy could be explained by the hypothesis that histone acetylation per se does not directly contribute to gene activation; instead, it is likely that globin gene activation is regulated by chromatin loop formation which is modulated by histone acetylation ( 26 ).
|
16914453_p22
|
16914453
|
DISCUSSION
| 4.294115 |
biomedical
|
Study
|
[
0.9995017051696777,
0.0002665040083229542,
0.00023169930500444025
] |
[
0.9991692304611206,
0.00022832915419712663,
0.0005225971108302474,
0.00007982238457771018
] |
en
| 0.999999 |
The results in this study suggest that each individual base in the 9 base zinc-finger binding motif has a distinct effect on γ gene expression. The CC→TG γ CACCC mutation disrupted protein binding and abolished γ-gene expression in adult erythropoiesis. The ‘down’ A→C mutation at −140γ does not seem to alter the type of binding proteins, but does increase the protein binding affinity in the major retarded band. The replacement of human γ CACCC box with human ɛ or galago γ CACCC box conferred to the γ gene an embryonic expression pattern ( 22 ). As seen on gel shift assay, all three CACCC boxes had higher binding affinity in comparison to the wild-type γ CACCC box. Based on these observations, we speculate that a change in binding affinity of the CACCC motif, not necessarily in the type of the binding proteins, could result in an alteration in expression pattern. This conclusion is consistent with the hypothesis that the combination of varying affinity with varying concentration is important in lineage-specific expression of the chicken ρ-globin gene ( 27 ). It should be pointed out that the 9 base sequence of the zinc-finger motif in the A→C mutated γ CACCC box is identical to the human ɛ CACCC box. However, the phenotypes of these two CACCC boxes were different, suggesting that nucleotides beyond the 9 base motif may also have a role in determining the binding property of a zinc-finger protein in vivo .
|
16914453_p23
|
16914453
|
DISCUSSION
| 4.313991 |
biomedical
|
Study
|
[
0.9994010925292969,
0.0003632431908044964,
0.0002356673066969961
] |
[
0.9992581009864807,
0.0002662800543475896,
0.00037310764309950173,
0.00010254043445456773
] |
en
| 0.999996 |
This study was designed to assess the transcriptional potential of the γ-gene promoter in different developmental stages, rather than to delineate the developmental regulation of the gene. Knowledge of transcriptional potential is essential for examining the function of the γ-gene promoter in the developmental regulation in the context of the entire locus. For instance, the finding that the CACCC box-less γ globin gene has a complete transcriptional potential in embryonic erythroid cells allow us to determine whether the CACCC box is required for loop formation between the LCR and the γ gene. The CACCC mutation can be introduced into the γ-gene promoter in a βYAC construct. If the CACCC box-less γ globin gene is expressed in the YAC construct, the result could suggest that the LCR enhancing activity is delivered to the gene via loop formation, and the CACCC box is not required for the event. If the mutated γ gene is not expressed, the most likely explanation will be that the CACCC box mutation results in a failure of loop formation between the LCR and the gene.
|
16914453_p24
|
16914453
|
DISCUSSION
| 4.198289 |
biomedical
|
Study
|
[
0.9994617104530334,
0.00029524185811169446,
0.00024304213002324104
] |
[
0.9994240999221802,
0.0002823424292728305,
0.000225749216042459,
0.00006777703674742952
] |
en
| 0.999997 |
Trypanosomatids are a group of exclusively parasitic kinetoplastid protozoa, which are responsible for several major human diseases. The most notable of these are sleeping sickness and South American Chagas' disease, caused by Trypanosoma spp., and the different forms of leishmaniasis, caused by Leishmania spp. Sleeping sickness is endemic in certain regions of Sub-Saharan Africa that encompass 36 countries and 60 million people. It is estimated that 300–500 thousand people are infected and 40 000 die every year of this disease. According to the World Health Organization, Chagas' disease currently affects 16–18 million people, particularly in the South America. Leishmaniasis adds another 12 million people living in 88 different countries ( 1 – 3 ).
|
16914442_p0
|
16914442
|
INTRODUCTION
| 4.00096 |
biomedical
|
Review
|
[
0.9988258481025696,
0.0004005874798167497,
0.0007735512335784733
] |
[
0.10504425317049026,
0.03320857882499695,
0.8610283136367798,
0.0007189129828475416
] |
en
| 0.999997 |
Recently, significant efforts have been placed on genome sequencing and annotation of both Trypanosoma and Leishmania , and several completely sequenced genomes of these organisms are currently available ( 4 – 6 ). Correct genome annotation and understanding of protein functions in these organisms are considered crucial for drug development and disease prevention ( 7 , 8 ). However, the use of existing annotation tools did not result in identification of genes coding for selenocysteine (Sec)-containing proteins because Sec, the 21st naturally occurring amino acid in the genetic code, is encoded by UGA, one of three signals that terminate protein synthesis ( 9 , 10 ). Leishmania major , Trypanosoma cruzi and Trypanosoma brucei were reported to contain a gene coding for a homolog of selenophosphate synthetase, an enzyme that generates selenophosphate, a selenium donor compound used for biosynthesis of Sec ( 11 ). However, whether this protein is functional in L.major is not known, and in addition, selenophosphate synthetase is also involved in pathways other than Sec biosynthesis ( 12 ). Thus, whether Leishmania or other Kinetoplastida utilize Sec remains unknown.
|
16914442_p1
|
16914442
|
INTRODUCTION
| 4.326339 |
biomedical
|
Study
|
[
0.9995276927947998,
0.0002166047052014619,
0.0002557226689532399
] |
[
0.9955617189407349,
0.0003404302697163075,
0.004003733396530151,
0.00009418823901796713
] |
en
| 0.999997 |
Sec is inserted into nascent polypeptides with the help of an RNA structure, designated Sec insertion sequence (SECIS) element ( 9 ). We previously reported that SECIS elements in closely related species show a significant level of homology and that evolutionary criteria could be applied to carry out computational searches with increased specificity for these structures ( 13 ). Since multiple completely sequenced Kinetoplastida genomes are available, simultaneous analysis of these genomes could help identify selenoprotein genes encoded in these organisms.
|
16914442_p2
|
16914442
|
INTRODUCTION
| 4.124803 |
biomedical
|
Study
|
[
0.9995322227478027,
0.00014963597641326487,
0.00031808321364223957
] |
[
0.9989639520645142,
0.0005694117280654609,
0.0004095938929822296,
0.00005712349229725078
] |
en
| 0.999995 |
Identification of selenoprotein genes in Kinetoplastida is also interesting from an evolutionary point of view: although selenoproteomes have been previously identified in the three domains of life, lower eukaryotes show variable dependence on selenium, with yeast and higher plants lacking selenoproteins ( 14 ). Few attempts have been made to explore a larger protozoan community ( 15 , 16 ). Thus, information about similarities and differences among protozoan selenoproteomes may provide valuable insights into evolution of selenium utilization.
|
16914442_p3
|
16914442
|
INTRODUCTION
| 4.075912 |
biomedical
|
Study
|
[
0.9995597004890442,
0.00014615850523114204,
0.0002940593403764069
] |
[
0.9967913031578064,
0.0006920560845173895,
0.002435382455587387,
0.00008121153950924054
] |
en
| 0.999996 |
In this study, we carried out bioinformatics analyses and identified three selenoprotein genes in Kinetoplastida. Two of them correspond to already known SelK and SelT families, while the third selenoprotein showed no homology to known proteins and thus represents a new selenoprotein family. Metabolic labeling of cells with 75 Se and auranofin inhibition studies supports these in silico findings. These data are discussed with respect to the dependence of Kinetoplastida on selenium.
|
16914442_p4
|
16914442
|
INTRODUCTION
| 4.122851 |
biomedical
|
Study
|
[
0.9995230436325073,
0.00024621005286462605,
0.00023081501421984285
] |
[
0.9994580149650574,
0.00018868432380259037,
0.0002948532928712666,
0.00005843139297212474
] |
en
| 0.999998 |
Nucleotide sequences of Trypanosoma congolense , T.cruzi , Trypanosoma vivax , Trypanosoma gambiense , Trypanosoma brucei brucei , L.major , Leishmania infantum and Leishmania braziliensis genomes, as well as predicted proteins sequences, were downloaded from The Wellcome Trust Sanger Institute ( ). SECISearch 2.19 ( 14 ) was used for identification of SECIS elements. FASTA package ( 17 ) and BLAST were used for similarity search. An online version of MFOLD version 3.2 ( 18 ) was used for RNA secondary structure prediction and preliminary analysis of SECIS-like structures. ClustalX was used for calculation of distances between each pair of sequences used to construct the phylogenetic tree.
|
16914442_p5
|
16914442
|
Databases and programs
| 4.144082 |
biomedical
|
Study
|
[
0.9996694326400757,
0.00017720702453516424,
0.00015333284682128578
] |
[
0.998829185962677,
0.00035516510251909494,
0.0007386135403066874,
0.00007708142948104069
] |
en
| 0.999998 |
In the search for homologs of known selenoprotein, query sequences were represented by Chlamydomonas MsrA ( 19 , 20 ), four Plasmodium falciparum selenoproteins ( 16 ), Gallus gallus SelU ( 21 ), protein disulfide isomerase from Emiliania huxleyi ( 22 ) and the full set of human selenoproteins ( 14 ). A stand-alone version of TBLASTN was used to detect nucleotide sequences corresponding to known selenoprotein families. Downstream regions of detected sequences were analyzed for presence of SECIS elements with SECISearch. In addition, nucleotide sequences were analyzed with MFOLD ( 18 ) to identify SECIS-like structures. All SECIS-like structures were screened for compliance with elements of the current SECIS consensus model (e.g. a non-Watson–Crick quartet in the SECIS core and unpaired AA, AG or CC nucleotides in the apical loop).
|
16914442_p6
|
16914442
|
Identification of distant homologs of known selenoprotein genes
| 4.168028 |
biomedical
|
Study
|
[
0.9995377063751221,
0.00023012254678178579,
0.000232177902944386
] |
[
0.9992883801460266,
0.000198710011318326,
0.0004549903969746083,
0.000058059507864527404
] |
en
| 0.999997 |
The default pattern of SECISearch was modified to accommodate Trypanosoma SECIS elements identified with the loose pattern of SECISearch and those not detectable by SECISearch but identified by manual searches with MFOLD. The modifications were as follows: (i) the threshold of the free energy of the overall structure was −11.5 kcal/mol, (ii) the minimum length of the stem was 10 bp and (iii) the apical loop was 3–17 nt. Genomic sequences of Kinetoplastida were searched using this modified version of SECISearch. Details of the procedures have been previously described ( 14 , 16 ). Briefly, nucleotide sequences were identified in the genome that meet primary and secondary sequence/structure requirements, satisfy free energy criteria and pass additional structural filters.
|
16914442_p7
|
16914442
|
Searches for SECIS elements
| 4.163335 |
biomedical
|
Study
|
[
0.9995418787002563,
0.00019786172197200358,
0.0002602463064249605
] |
[
0.9992703795433044,
0.00034966415842063725,
0.00032295132405124605,
0.00005706526280846447
] |
en
| 0.999998 |
All SECIS candidates were analyzed for the presence of at least one homolog in other Kinetoplastida species by searches against a database containing SECIS candidates from other Kinetoplastida species. To align SECIS sequences, we used FASTA with an E -value of 1 × 10 −8 . Regions upstream of SECIS elements were further analyzed for occurrence of open reading frames (ORFs). An additional requirement was the presence of at least two homologous ORFs in Kinetoplastida. Candidates, in which SECIS elements and ORFs were on different DNA strands, were filtered out.
|
16914442_p8
|
16914442
|
Searches for SECIS elements
| 4.105966 |
biomedical
|
Study
|
[
0.9994694590568542,
0.00024790308088995516,
0.00028265590663067997
] |
[
0.9993659853935242,
0.0002557066618464887,
0.000320882216328755,
0.0000574506884731818
] |
en
| 0.999996 |
For the auranofin experiments, culture-adapted bloodstream and procyclic T.brucei of the cell line 449 [descendants of strain Lister 427 ( 23 ) and stably transfected with pHD449 encoding the tetracycline repressor ( 24 )] were used. Bloodstream T.brucei brucei was grown at 37°C in a humidified atmosphere with 5% CO 2 in HMI-9 medium supplemented with 1.5 mM cysteine, 0.0014% (v/v) β-mercaptoethanol, 10% heat-inactivated fetal calf serum (FCS) (v/v), 50 U/ml penicillin, 50 μg/ml streptomycin and 0.2 μg/ml phleomycin. Procyclic T.brucei were grown in MEM-Pros medium (Biochrom) supplemented with 7.5 μg/ml hemin, 10% heat-inactivated FCS (v/v), 50 U/ml penicillin, 50 μg/ml streptomycin and 0.5 μg/ml phleomycin at 27°C.
|
16914442_p9
|
16914442
|
Cultivation of bloodstream and procyclic T.brucei brucei
| 4.144372 |
biomedical
|
Study
|
[
0.9995773434638977,
0.0002261782210553065,
0.0001964596303878352
] |
[
0.9990302324295044,
0.0006244551623240113,
0.00027222649077884853,
0.00007307268970180303
] |
en
| 0.999997 |
Auranofin, a highly specific inhibitor of several selenoenzymes ( 25 , 26 ) was tested as an inhibitor of trypanosomal growth. A total of 1 × 10 5 bloodstream trypanosomes per ml or 2 × 10 5 procyclic parasites per ml were cultured as described above in the presence of 1 nM to 10 μM of auranofin dissolved in dimethyl sulfoxide (DMSO). To rule out that DMSO by itself might influence growth of cells, an additional control experiment without DMSO was carried out in parallel. A sample without auranofin but with an identical volume of DMSO served as an additional control. After 18 h, the cells were counted in a Neubauer chamber. No growth difference could be detected between these two negative controls. All experiments were carried out in duplicate.
|
16914442_p10
|
16914442
|
Auranofin inhibition studies
| 4.087972 |
biomedical
|
Study
|
[
0.9994606375694275,
0.00030798441730439663,
0.00023138859251048416
] |
[
0.9994392991065979,
0.00020791814313270152,
0.0002949858899228275,
0.000057817622291622683
] |
en
| 0.999996 |
T.cruzi (Tulahuen-2 strain) epimastigotes (proliferative and extracellular stage) were cultured at 28°C in BHI medium [33 mg/ml brain-heart infusion (Difco)] supplemented with 3 mg/ml tryptose, 20 μg/ml hemin, 5 mM KCl and 25 mM sodium phosphate, complement-inactivated 10% fetal bovine serum (v/v), 1.7 mM glucose, 200 μg/ml streptomycin sulfate and 200 U/ml penicillin at pH 7.3. A total of 100 ml exponential-phase parasites (2 × 10 7 cells per ml) were harvested by centrifugation at 800 g (Sorvall RC5Cplus, rotor F21S FiberLite) and washed twice with DMEM (Dulbecco's Modified Eagle Medium, SIGMA), without fetal bovine serum but supplemented with 1.5 mM l -glutamine, 5.6 mM glucose, 45 mM sodium bicarbonate, 200 μg/ml streptomycin sulfate and 200 U/ml penicillin. The collected parasites were resuspended in 5 ml of DMEM and cultured for 40 h at 28°C in the presence of 400 μCi of 75 Se provided as 5 μM [ 75 Se]selenite (University of Missouri Research Reactor). After 40 h, cells were harvested by centrifugation, washed twice, resuspended in 500 μl of phosphate-buffered saline (PBS) containing 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1 mM (2 S ,3 S )-3-( N- {( S )-1-[ N- (4-guanidinobutyl) carbamoyl] 3-methylbutyl}carbamoyl)oxirane-2-carboxylic acid (E-64), and sonicated. A total of 10–20 μg of protein from parasite cell homogenates were subjected to 10% SDS–PAGE under reducing conditions and transferred onto a polyvinylidine difluoride (PVDF) membrane. Proteins were stained with Coomassie blue and radioactivity was visualized by autoradiography using a PhosphorImager (Fuji).
|
16914442_p11
|
16914442
|
Metabolic labeling with 75 Se
| 4.248852 |
biomedical
|
Study
|
[
0.9994359612464905,
0.00034538970794528723,
0.0002185730991186574
] |
[
0.9986646175384521,
0.0007966996636241674,
0.00043680379167199135,
0.00010186197323491797
] |
en
| 0.999998 |
To identify selenoprotein genes in Trypanosoma and Leishmania , we initially searched available sequenced genomes of these organisms for occurrence of homologs of known selenoprotein genes with TBLASTN. Two selenoprotein families were found in several Kinetoplastida genomes, including homologs of human SelK (accession no. Q9Y6D0) and SelT .
|
16914442_p12
|
16914442
|
RESULTS AND DISCUSSION
| 4.10791 |
biomedical
|
Study
|
[
0.999578058719635,
0.0002007611037697643,
0.00022112404985819012
] |
[
0.9991641044616699,
0.000508989323861897,
0.00026437704218551517,
0.0000624522435828112
] |
en
| 0.999996 |
Alignments of SelK SECIS elements and protein sequences are shown in Figures 1 and 2 . All SECIS elements in the SelK family could be found using the default pattern of SECISearch. In addition, both SECIS elements and protein sequences were highly conserved among Trypanosomas and Leishmania. Thus, the SelK SECIS elements found in Kinetoplastida sequences fit very well the eukaryotic SECIS consensus model.
|
16914442_p13
|
16914442
|
RESULTS AND DISCUSSION
| 4.171071 |
biomedical
|
Study
|
[
0.9994507431983948,
0.00022181522217579186,
0.0003274473128840327
] |
[
0.9991376399993896,
0.00047062226803973317,
0.0003220638900529593,
0.00006961427425267175
] |
en
| 0.999996 |
In contrast, SECIS elements in SelT genes differed from the typical SECIS structure. While SelT protein sequences showed high conservation rate among Kinetoplastida , SECIS elements were much less conserved, and identification of several SelT SECIS elements posed a challenge even with the loose pattern of SECISearch.
|
16914442_p14
|
16914442
|
RESULTS AND DISCUSSION
| 3.793993 |
biomedical
|
Study
|
[
0.99880051612854,
0.00017275473510380834,
0.0010266659082844853
] |
[
0.9951966404914856,
0.004355927929282188,
0.00034264393616467714,
0.00010478302283445373
] |
en
| 0.999997 |
To adjust SECISearch for identification of Trypanosoma SECIS elements, we developed a modified version of the program (as described in Materials and Methods), which significantly improved specificity of the searches. Application of this program resulted in the detection of all SelT SECIS elements except that in the T.cruzi SelT gene. Interestingly, predicted SECIS elements in L.major and L.infantum SelT genes drastically differed from their T.gambiense , T.congolense and T.vivax counterparts.
|
16914442_p15
|
16914442
|
RESULTS AND DISCUSSION
| 4.136018 |
biomedical
|
Study
|
[
0.9995898604393005,
0.00016886966477613896,
0.00024132180260494351
] |
[
0.9991708993911743,
0.000495188229251653,
0.00027498992858454585,
0.000058941128372680396
] |
en
| 0.999998 |
We further applied the modified version of SECISearch to analyze entire genomes and genome survey sequences of T.congolense , T.cruzi , T.vivax , T.gambiense , T.brucei brucei , L.major , L.infantum and L.braziliensis . An additional requirement was the presence of SECIS homologs in other Kinetoplastida (cut-off value of 1 × 10 −8 as determined from FASTA alignments of Trypanosoma and Leishmania SECIS elements in SelK and SelT genes). However, L.major , L.infantum and L.brasiliensis genomes exhibited very high sequence similarity; therefore, only L.major was included in the searches. Upstream regions of SECIS element candidates were analyzed for the presence of at least one homolog in other Kinetoplastida species. Predicted ORFs were then searched against NCBI non-redundant protein database, as well as against predicted Kinetoplastida proteins.
|
16914442_p16
|
16914442
|
RESULTS AND DISCUSSION
| 4.179502 |
biomedical
|
Study
|
[
0.9995429515838623,
0.0002545717288739979,
0.0002025390276685357
] |
[
0.9993271827697754,
0.00019663499551825225,
0.0004055564058944583,
0.0000706543869455345
] |
en
| 0.999995 |
This analysis identified six homologous groups of candidates, including homologs of SelK and SelT, as well as a new selenoprotein family designated SelTryp ( Table 1 ). Two other candidates were filtered out because SECIS elements and ORFs were on different DNA strands, and the last SECIS candidate corresponded to a predicted ORF with no suitable in-frame TGA triplet.
|
16914442_p17
|
16914442
|
RESULTS AND DISCUSSION
| 4.098315 |
biomedical
|
Study
|
[
0.9995124340057373,
0.00020129601762164384,
0.00028633407782763243
] |
[
0.999264657497406,
0.0004738324787467718,
0.00019455523579381406,
0.00006695118645438924
] |
en
| 0.999996 |
SelTryp SECIS elements, their alignments, and alignments of the corresponding selenoproteins are shown in Figures 5 and 6 . Like SelT structures, SelTryp SECIS elements were conserved within Trypanosoma and within Leishmania , but little conservation was detected between Trypanosoma and Leishmania SelTryp SECIS elements .
|
16914442_p18
|
16914442
|
RESULTS AND DISCUSSION
| 3.985978 |
biomedical
|
Study
|
[
0.9995654225349426,
0.00015117114526219666,
0.0002833477919921279
] |
[
0.9983890056610107,
0.0011667396174743772,
0.00036539402208290994,
0.0000789379482739605
] |
en
| 0.999996 |
In the SelTryp ORF, Sec was present in the C-terminal region, within a conserved C-terminal peptide, SI(V)I(V) CI(V)SU PR (U is Sec). Although in known selenoproteins Sec is most often found in loops located between secondary structures, the C-terminal location is also common to eukaryotic selenoproteins (e.g. thioredoxin reductase, SelK, SelS and SelO). In SelTryp, Sec is present within a CxxU motif, which is often found in selenoproteins that carry our redox function through reversible formation of a selenenylsulfide bond. This observation suggests a redox function for the CxxU motif in SelTryp.
|
16914442_p19
|
16914442
|
RESULTS AND DISCUSSION
| 4.411433 |
biomedical
|
Study
|
[
0.9993616938591003,
0.0002880573447328061,
0.00035024178214371204
] |
[
0.9980894923210144,
0.0013514378806576133,
0.00040070965769700706,
0.00015830766642466187
] |
en
| 0.999998 |
Analysis of SelTryp sequences revealed distant homology to a rhodanese-like protein from Thermobifida fusca YX . Two rhodanese homology domains (RHOD) could be seen in SelTryp by CD-Search ( 27 ) in the region spanning 500–585 and 621–774 amino acids. An additional analysis of amino acid sequences using SMART ( 28 ), AnDom ( 29 ) and PROSITE ( 30 ) predicted the occurrence of a metallo-β-lactamase fold . The presence of conserved cysteines in the rhodanese domains within a 6 amino acid active site loop (CXGGXR) suggested that this protein belonged to a YceA subfamily ( 31 ). In addition, the use of DisEMBL ( 32 ) revealed the lack of secondary structures in the C-terminal region of SelTryp, suggesting a flexible C-terminal Sec-containing tail.
|
16914442_p20
|
16914442
|
RESULTS AND DISCUSSION
| 4.373508 |
biomedical
|
Study
|
[
0.9994271993637085,
0.0003068676742259413,
0.0002658842713572085
] |
[
0.9991081357002258,
0.0003237443452235311,
0.0004583566915243864,
0.00010973222379107028
] |
en
| 0.999996 |
The N-terminal sequences of SelTryp belong to a metallo-β-lactamase superfamily of proteins and are followed with rhodanese domains. Proteins with the metallo-β-lactamase fold catalyze a wide variety of reactions, partly because this fold allows selectivity for different metals. For example, hydrolytic metallo-β-lactamase proteins mostly bind zinc, redox-active rubredoxin:oxygen-oxidoreductases contain a di-iron cluster, and glyoxalases II (thiolesterases) contain iron, manganese or zinc ( 33 ).
|
16914442_p21
|
16914442
|
RESULTS AND DISCUSSION
| 4.357062 |
biomedical
|
Study
|
[
0.9995736479759216,
0.00016416683502029628,
0.0002621580206323415
] |
[
0.99265056848526,
0.004945109598338604,
0.002201278228312731,
0.00020311534171923995
] |
en
| 0.999997 |
Subcellular localization predictions using iPSORT [( 34 ), ], TargetP 1.1 [( 35 ), ] and PredictProtein [( 36 ), ) suggested a mitochondrial localization of SelTryp, whereas the SelT sequence contains a potential export signal. In good agreement with its human homolog, the Kinetoplastida SelK has a predicted transmembrane motif.
|
16914442_p22
|
16914442
|
RESULTS AND DISCUSSION
| 4.1845 |
biomedical
|
Study
|
[
0.9995878338813782,
0.0001410629047313705,
0.0002711202541831881
] |
[
0.9984288811683655,
0.0009838786209002137,
0.0004985393024981022,
0.00008867996803019196
] |
en
| 0.999996 |
All selenoprotein-coding genes that were found in Leishmania and Trypanosoma lacked introns. In Leishmania selenoproteins, the distance from Sec-encoding UGA codons to SECIS elements was between 800 bp (SelTryp) and 1100 bp (SelK). In Trypanosoma selenoprotein genes, this distance was more variable: ∼200 bp for SelK and SelTryp, and 850 bp for SelT.
|
16914442_p23
|
16914442
|
RESULTS AND DISCUSSION
| 4.210532 |
biomedical
|
Study
|
[
0.9995748400688171,
0.00016299699200317264,
0.00026214407989755273
] |
[
0.998412013053894,
0.001041194424033165,
0.00046608035336248577,
0.00008070933108683676
] |
en
| 0.999996 |
Analysis of selenoprotein ORFs against annotated Leishmania and Trypanosoma genomes revealed that SelK genes were not annotated at all, except for the T.cruzi SelK gene, for which a wrong ORF was predicted. SelTryp genes were misannotated because the in-frame UGA codons were interpreted as stop signals. SelT genes were split into two parts. One part corresponded to the N-terminal regions of the proteins and was predicted to terminate at the Sec-encoding UGA codons, and the second was predicted to initiate from an AUG codon, which corresponded to the internal methionine downstream of the Sec UGA codon, continuing until the true stop signal.
|
16914442_p24
|
16914442
|
RESULTS AND DISCUSSION
| 4.329948 |
biomedical
|
Study
|
[
0.9994650483131409,
0.00028709156322292984,
0.00024786018184386194
] |
[
0.9988671541213989,
0.000647093984298408,
0.000367420056136325,
0.00011835127224912867
] |
en
| 0.999996 |
In addition to protein components of the Sec insertion machinery (selenophosphate synthetase, Sec tRNA-specific elongation factor), Sec tRNA genes were identified. Although tRNAscan-SE used with default settings ( 37 ) failed to recognize Sec tRNA genes in L . major , the use of ARAGORN ( 38 ) identified Sec tRNAs in Trypanosoma and Leishmania genomes. The predicted Sec tRNAs were then successfully verified using COVE with a Sec tRNA profile. All Sec tRNAs in Kinetoplastida could also be found with a tool adapted for unusual tRNAs ( 39 ). All Sec insertion machinery genes and selenoprotein genes were located either on different chromosomes or, when on the same chromosome, they were distant from each other. Thus, although Kinetoplastida form operon-like structures, the Sec insertion trait genes are spread throughout the parasite genomes.
|
16914442_p25
|
16914442
|
RESULTS AND DISCUSSION
| 4.244046 |
biomedical
|
Study
|
[
0.9995225667953491,
0.00022505861124955118,
0.000252308149356395
] |
[
0.9992700219154358,
0.00029127279412932694,
0.00037029167287983,
0.00006835973908891901
] |
en
| 0.999997 |
As a preliminary test to verify that these sequences are indeed expressed as predicted we exploited two peculiarities of Trypanosomes. First, mature T.brucei brucei mRNA molecules share a common 5′ sequence (the so called spliced leader, SL). Thus, a common forward primer can be used for PCR on a T.brucei brucei cDNA preparation and subsequent sequencing allows verification that a predicted start codon is correct and to rule out that our sequence is a part of a larger one. Second, as trypanosomal protein expression is primarily regulated at the transcriptional level, the detection of the respective mRNA (via cDNA) is essentially a proof that the respective protein is expressed. Using this approach, we were able to identify unequivocally the respective mRNA of SelT and SelK in T.brucei brucei (data not shown).
|
16914442_p26
|
16914442
|
RESULTS AND DISCUSSION
| 4.158547 |
biomedical
|
Study
|
[
0.9994589686393738,
0.0002682149061001837,
0.0002728388353716582
] |
[
0.9993662238121033,
0.0003586267994251102,
0.00020679834415204823,
0.00006833254883531481
] |
en
| 0.999996 |
To directly test if selenium is inserted into Trypanosoma proteins, we metabolically labeled Trypanosoma cells with 75 Se. Following SDS–PAGE, the 75 Se profile was visualized with a PhosphorImager. A major 75 Se-containing high-molecular weight band was detected at the top of the gel . This 75 Se species was insoluble , but selenium could be partially released by treatment with urea and high concentrations of reducing agents (data not shown). This band did not correspond to the three selenoproteins, and in fact this form of selenium has not been previously observed in other species. Determination of the nature of the high-molecular weight selenium species should await further studies.
|
16914442_p27
|
16914442
|
RESULTS AND DISCUSSION
| 4.111844 |
biomedical
|
Study
|
[
0.9995242357254028,
0.00022523569350596517,
0.0002506353484932333
] |
[
0.9994338154792786,
0.00026357246679253876,
0.0002487371093593538,
0.0000538669410161674
] |
en
| 0.999996 |
The soluble fraction of Trypanosoma cell extracts had little 75 Se . However, longer exposure to a PhosphorImager screen revealed a 10 kDa band that migrated in accord with the predicted molecular mass of SelK . In addition, minor bands that were labeled with 75 Se could be detected, but they corresponded to a protein profile detected by Coomassie blue staining . These additional bands probably derived from non-specific labeling of proteins with selenium wherein this trace element entered sulfur pathways and was inserted in place of sulfur in methionine and cysteine residues. However, the candidate radioactive SelK band had no corresponding protein band, further suggesting that this was a specific selenoprotein band. These data show that selenoproteins are expressed in Trypanosoma cells in the life-cycle stage at low level, and that only some selenoproteins could be visualized by metabolic 75 Se labeling. Whereas SelT and SelTryp could not be detected with 75 Se, specific 75 Se insertion into 10 kDa and high-molecular weight bands verified our prediction of the use of selenium by Trypanosoma cells.
|
16914442_p28
|
16914442
|
RESULTS AND DISCUSSION
| 4.333076 |
biomedical
|
Study
|
[
0.9994693398475647,
0.00030259607592597604,
0.00022807256027590483
] |
[
0.9992057681083679,
0.0002785953402053565,
0.00043411459773778915,
0.00008147815970005468
] |
en
| 0.999996 |
Gold(I) compounds, such as auranofin, are highly specific inhibitors of several eukaryotic selenoenzymes. We therefore studied the impact of auranofin on the growth of T.brucei brucei . As shown in Figure 8 , this compound was highly toxic for bloodstream and procyclic stages of the parasite with IC 50 values in the lower nanomolar range. This renders auranofin a highly interesting drug candidate per se . It should be noted that the growth medium contains significant amounts of plasma proteins. As auranofin is preferentially bound to plasma proteins (∼60%), it can be assumed that the active (i.e. free) drug concentration in our experiment was even lower ( 40 ). In this context, it is important to stress that the three selenoproteins contain putative redox centers (CxxU in the case of SelT and SelTryp, and CxxxU in SelK), and that impaired redox balance may influence Kinetoplastid infections ( 41 ). Yet, further studies are required to verify the selenium dependence and determine the concentration of this trace element that is required for viability of the parasites in different developmental stages.
|
16914442_p29
|
16914442
|
RESULTS AND DISCUSSION
| 4.233233 |
biomedical
|
Study
|
[
0.9994958639144897,
0.00030739401699975133,
0.0001966599520528689
] |
[
0.9993118047714233,
0.00020227389177307487,
0.0004102203238289803,
0.00007565339183202013
] |
en
| 0.999998 |
To examine the evolutionary history of the Sec trait in Kinetoplastida, we constructed a phylogenic tree for Sec tRNA . Trypanosoma sequences clustered with other eukaryotes, suggesting a common origin of the Sec insertion system. In the tree, Trypanosoma Sec tRNAs formed a cluster with animal Sec tRNAs and Chlamydomonas Sec tRNA. This cluster was separated from the plasmodial cluster. Together with the finding of a eukaryotic Sec-specific elongation factor, these data suggest that the Sec insertion system is Kinetoplastida is similar to the previously characterized eukaryotic Sec insertion systems. Nevertheless, identification of a protein specific for this group of organisms (SelTryp) highlights the fact that low eukaryotes may possess novel selenoproteins. One recent study revealed four such proteins in Plasmodia ( 16 ). With an ever increasing pace at which new genome sequences become completed, further computational analyses should reveal yet additional selenoproteins, and with them new pathways of selenium utilization in biology.
|
16914442_p30
|
16914442
|
RESULTS AND DISCUSSION
| 4.283329 |
biomedical
|
Study
|
[
0.9994636178016663,
0.0002868021547328681,
0.0002496492234058678
] |
[
0.9991052746772766,
0.00025285666924901307,
0.0005700397887267172,
0.00007183387060649693
] |
en
| 0.999997 |
In conclusion, we carried out an in silico analysis of all available sequenced Kinetoplastida genomes for the presence of selenoprotein genes. By computationally predicting SECIS elements, we characterized the Trypanosoma and Leishmania selenoproteomes, which consist of three selenoproteins. Among them, SelT and SelK were distant homologs of previously identified mammalian selenoproteins. A new selenoprotein, SelTryp, was also discovered. This selenoprotein has two rhodanese and one rubredoxin:oxygen oxidoreductase domains and appears to be a Kinetoplastida-specific multidomain redox protein of unknown function. All selenoprotein genes were previously misannotated in sequence databases. Metabolic labeling of Trypanosoma cells with 75 Se revealed specific insertion of this radioisotope into a defined set of proteins, and in addition, Trypanosoma cells were found to be sensitive to a gold(I) compound, auranofin, which specifically targets selenoproteins. These findings, together with the presence of the Sec-decoding trait in Kinetoplastida genomes suggest that these organisms utilize selenium and depend on this trace element, and that this dependence is likely due to the occurrence of at least three selenoproteins in these organisms. The absence of SelTryp homologs (either Sec or Cys forms) in the human host may also be relevant to drug development: selective inhibition of this selenoprotein might lead to new drugs to treat typanosomatid infections. Finally, these findings highlight the fact that lower eukaryotes evolved unique selenoproteomes, whose analysis should suggest new uses of the trace element selenium in biology.
|
16914442_p31
|
16914442
|
RESULTS AND DISCUSSION
| 4.499388 |
biomedical
|
Study
|
[
0.9992913007736206,
0.0004960065125487745,
0.00021269332501105964
] |
[
0.9982900023460388,
0.0003601211355999112,
0.0011747897369787097,
0.00017509875760879368
] |
en
| 0.999995 |
The immune response against pathogenic microorganisms involves two components: a rapid, antigen nonspecific, innate response and a delayed, acquired response specific for the antigens displayed by the invading microbe. Studies using the intracellular bacterium Listeria monocytogenes (LM) have been useful in establishing the kinetics and mechanisms of both the innate and adaptive immune responses against intracellular pathogens in general. The innate immune response to LM is a complex network involving multiple cell types, cytokines, and bactericidal effector mechanisms ( 1 , 2 ). The adaptive immune response to LM is T cell–mediated and required for complete resolution of the bacterium as evidenced by studies using T cell–deficient mice ( 3 ).
|
14623912_p0
|
14623912
|
Introduction
| 4.29565 |
biomedical
|
Study
|
[
0.9995906949043274,
0.00024292958551086485,
0.00016632753249723464
] |
[
0.9966068267822266,
0.00046038065920583904,
0.0028401988092809916,
0.00009265259723179042
] |
en
| 0.999996 |
In the experimental murine model of LM infection, circulating monocytes and resident macrophages, such as Kupffer cells, play an important role in innate immunity by ingesting and destroying LM ( 1 ). The generation of reactive nitrogen and oxygen intermediates by infected macrophages and neutrophils results in direct killing of LM ( 4 – 6 ), although mechanisms independent of these intermediates also can play a role in host resistance ( 7 – 9 ). In support of the role of phagocytic cells and their effector functions, depletion of macrophages ( 10 ) or neutrophils ( 11 ) results in impaired innate resistance against LM.
|
14623912_p1
|
14623912
|
Introduction
| 4.229906 |
biomedical
|
Study
|
[
0.999581515789032,
0.0002677578304428607,
0.00015078137221280485
] |
[
0.997832715511322,
0.0003560439217835665,
0.0017161702271550894,
0.00009501982276560739
] |
en
| 0.999996 |
Multiple cells of the immune system play a role in the innate response by reducing the bacterial load directly and secreting cytokines such as IL-1, IL-6, TNF-α, IFN-γ, and others. IL-1 is produced by LM-infected macrophages, and blockade of the IL-1 signaling pathway leads to an increased LM burden in mice ( 12 ). The mechanism involved in IL-1–mediated protection from LM infection is probably recruitment and activation of both neutrophils and macrophages ( 13 ). Likewise, IL-6 is rapidly secreted after LM infection ( 14 ). Removing this cytokine by depletion or gene targeting results in increased susceptibility to LM due to inefficient neutrophil recruitment ( 15 , 16 ). A recent report has shown increased LM susceptibility in lymphotoxin β receptor–deficient mice ( 17 ). Although TNF-α and IFN-γ production were not affected in these mice, nitric oxide production by macrophages was impaired.
|
14623912_p2
|
14623912
|
Introduction
| 4.330129 |
biomedical
|
Study
|
[
0.9995328187942505,
0.0002687106898520142,
0.0001984807604458183
] |
[
0.9988780617713928,
0.00022605729463975877,
0.0008060840191319585,
0.00008969943883130327
] |
en
| 0.999997 |
Studies using mice deficient in either TNF-α or its receptor identify this cytokine as being important in innate immunity to LM ( 18 , 19 ). TNF-α functions not only to induce IFN-γ production ( 20 ) but also to directly activate macrophages independent of IFN-γ ( 21 ). IL-12 and IL-18, produced mainly by activated macrophages, are both important mediators in the immune response against LM, and their primary function is to induce IFN-γ secretion from responding immune cells ( 20 – 23 ). However, an IFN-γ–independent role in controlling LM has also been proposed for IL-18 ( 24 ).
|
14623912_p3
|
14623912
|
Introduction
| 4.274728 |
biomedical
|
Study
|
[
0.9995618462562561,
0.00022488593822345138,
0.00021333462791517377
] |
[
0.9975889921188354,
0.0003199999046046287,
0.002011503092944622,
0.00007959003414725885
] |
en
| 0.999997 |
IFN-γ is produced by multiple cell types including NK cells ( 25 ), NK-T cells ( 26 ), macrophages ( 27 ), B cells ( 28 ), dendritic cells ( 29 ), and γδ T cells ( 30 ). Evidence that IFN-γ is critically important in the innate immune response comes from experiments using mice deficient in either the cytokine or its receptor. These animals rapidly succumb when infected with low doses of LM ( 31 , 32 ). SCID mice, which lack T and B lymphocytes, show increased susceptibility to LM when they are depleted of either IFN-γ or IL-12 ( 21 , 33 ). Providing IFN-γ to IL-12–depleted SCID mice can reverse this effect ( 21 ). One mechanism for the increased susceptibility of animals lacking the IFN-γ receptor, or presumably the cytokine itself, is impaired macrophage activity ( 34 ).
|
14623912_p4
|
14623912
|
Introduction
| 4.234048 |
biomedical
|
Study
|
[
0.9996364116668701,
0.0002075421070912853,
0.00015597630408592522
] |
[
0.998145580291748,
0.0003340310649946332,
0.001437587197870016,
0.00008275263826362789
] |
en
| 0.999998 |
It is generally thought that NK cells play an important role in the control of LM infection due to secretion of IFN-γ induced by IL-12 and IL-18 ( 25 ). In contrast, one study found that depleting NK cells led to decreased LM burdens in B6 mice ( 35 ). However, the IFN-γ–secreting activity of NK cells or any other cell type has never been directly tested for its ability to provide innate protection against LM infection. One cell type that can contribute to IFN-γ secretion during the innate immune response is CD8 + T cells. We and others have shown that CD44 hi CD8 + T cells respond to cytokines by secreting IFN-γ rapidly after infection with intracellular bacteria ( 36 , 37 ). Since this population of CD8 + T cells is as numerous as NK cells in normal mice, we directly tested their activity in vivo. This report demonstrates that memory CD8 + T cells provide protection against LM infection in IFN-γ–deficient animals in an antigen nonspecific manner.
|
14623912_p5
|
14623912
|
Introduction
| 4.261272 |
biomedical
|
Study
|
[
0.999452531337738,
0.0003223998937755823,
0.0002251149562653154
] |
[
0.9992921352386475,
0.00025963751249946654,
0.0003724008274730295,
0.0000758565729483962
] |
en
| 0.999997 |
C57BL/6J (B6), C57BL/6.PL-Thy1 a /Cy (B6.Thy1.1), B6.129S7- Ifng tm1Ts (IFN-γ −/− ), and OT-I TCR transgenic mice were bred and maintained at the University of Texas Southwestern Medical Center animal facility under the approval of the Institutional Animal Care and Use Committee.
|
14623912_p6
|
14623912
|
Mice.
| 3.190272 |
biomedical
|
Study
|
[
0.9987665414810181,
0.0003228182904422283,
0.0009107645601034164
] |
[
0.9044032692909241,
0.09340465068817139,
0.0015434412052854896,
0.000648646499030292
] |
en
| 0.999998 |
For infection of cell lines and mice, log phase cultures of LM 10403 serotype 1 or LM-expressing full-length ovalbumin protein (LM/OVA), provided by Dr. Hao Shen (University of Pennsylvania School of Medicine, Philadelphia, PA) ( 38 ), were washed twice and diluted in PBS to the desired concentration. LM or LM/OVA were injected in the lateral tail vein at the indicated dosage.
|
14623912_p7
|
14623912
|
Bacteria and Viruses.
| 4.048668 |
biomedical
|
Study
|
[
0.9996235370635986,
0.0002044497086899355,
0.00017196520639117807
] |
[
0.9970353841781616,
0.0023069430608302355,
0.0005498637328855693,
0.00010786918574012816
] |
en
| 0.999998 |
Vesicular stomatitis virus (VSV)–expressing full-length ovalbumin protein (VSV/OVA) was provided by Dr. Leo Lefrancois (University of Connecticut Health Center, Farmington, CT) ( 39 ). Vaccinia virus (VV)–expressing full-length ovalbumin protein (VV/OVA) was provided by Dr. Jack Bennink (National Institutes of Health, Bethesda, MD) ( 40 ). VSV/OVA and VV/OVA were injected in the lateral tail vein at a dosage of 2 × 10 6 PFU for a primary response.
|
14623912_p8
|
14623912
|
Bacteria and Viruses.
| 3.931685 |
biomedical
|
Study
|
[
0.9995293617248535,
0.00012801449338439852,
0.00034263156703673303
] |
[
0.9881159067153931,
0.010639178566634655,
0.001088021555915475,
0.00015682204684708267
] |
en
| 0.999997 |
The J774 macrophage line (H-2 d ) and the J774 macrophage line stably transfected with H-2K b (J774:K b ) were grown in complete DMEM (Life Technologies) supplemented with 10% FCS (Atlanta Biologicals). For overnight in vitro experiments, the culture of mouse splenocytes was performed in complete RPMI (Life Technologies) supplemented with 10% FCS and 10 ng/ml recombinant human IL-2 (provided by Dr. Michael Bennett, UTSW). In experiments designed to test the direct ex vivo activity of T cells, splenocytes were cultured for 3 h in complete RPMI supplemented with 10% FCS (without added cytokines or antigens). Recombinant murine IL-12 (5 ng/ml final concentration) and IL-18 (10 ng/ml final concentration) were from Peprotech Inc. Blocking antibodies against murine IL-12 and IL-18 (1 μg/ml final concentration) were purchased from Peprotech Inc. and Medical and Biological Laboratories Co., Ltd., respectively. Carboxyfluorescein diacetate, succinimidyl ester (CFSE) labeling of splenocytes was performed at a final concentration of 1 μM and was obtained from Molecular Probes. The OVA-derived peptide SIINFEKL was synthesized by the UTSW peptide synthesis facility.
|
14623912_p9
|
14623912
|
Cell Lines, Cell Culture, and Reagents.
| 4.15205 |
biomedical
|
Study
|
[
0.9996200799942017,
0.00021308474242687225,
0.00016686848539393395
] |
[
0.9986563920974731,
0.0009266362758353353,
0.00033829532912932336,
0.0000785647498560138
] |
en
| 0.999998 |
For cell staining experiments, the following antibodies from BD Biosciences were used: anti-CD8α (53–6.7), anti-CD44 (IM7), anti-CD90.2 (Thy1.2) (53–2.1), anti-CD94 (18d3), and anti–IFN-γ (XMG1.2). Secondary streptavidin-conjugated reagents were used to reveal biotinylated primary or secondary antibodies. Intracellular staining, data acquisition, and data analysis were performed as described previously ( 36 ). Staining for the presence of the IL-18Rα subunit was accomplished using an anti–IL-18Rα antibody (R&D Systems) followed by biotinylated anti–goat IgG (Jackson ImmunoResearch Laboratories), which was then revealed with streptavidin-conjugated PE. Identification of endogenous OVA-specific T cells was performed by first incubating the splenocytes with a blocking CD8α antibody (CT-CD8α) from Caltag Laboratories. After washing, the splenocytes were incubated at 4°C for 1 h with CD8α FITC and a K b -SIINFEKL tetramer coupled to PE that was purchased from the Protein Chemistry Core Laboratory at Baylor College of Medicine.
|
14623912_p10
|
14623912
|
Antibodies and Cell Staining.
| 4.144871 |
biomedical
|
Study
|
[
0.9995707869529724,
0.00024690444115549326,
0.0001823598431656137
] |
[
0.998336911201477,
0.001186271314509213,
0.0003845449537038803,
0.00009223284723702818
] |
en
| 0.999997 |
Red blood cell–depleted splenocytes were cultured in 24-well plates at a concentration of 3 × 10 6 /well. J774 or J774:K b macrophages cultured in antibiotic-free media were infected at a multiplicity of infection of 5:1 for 1 h. The macrophages were then washed three times with PBS and cultured for 3 h in complete DMEM supplemented with 10% FCS and gentamycin (100 μg/ml). The infected macrophages were then harvested and plated at 3 × 10 5 /well with the splenocytes.
|
14623912_p11
|
14623912
|
In Vitro Stimulations.
| 4.130834 |
biomedical
|
Study
|
[
0.9993622899055481,
0.0004360665625426918,
0.00020164312445558608
] |
[
0.9951730370521545,
0.00412791920825839,
0.0005243557970970869,
0.00017465029668528587
] |
en
| 0.999997 |
Splenocytes from OT-I TCR transgenic mice were passed over nylon wool columns to enrich for T cells. ∼3 × 10 6 cells were then injected i.v. into the lateral tail vein of B6.Thy1.1 recipient mice, rested for 1–3 d, and challenged with the indicated pathogen as described above. For the experiments analyzing OT-I T cells that were transferred and not challenged, 10 7 OT-I T cells were transferred into B6.Thy1.1 hosts, and the mice were killed 1–3 d later for analysis.
|
14623912_p12
|
14623912
|
T Cell Transfers and In Vivo Stimulations.
| 4.09632 |
biomedical
|
Study
|
[
0.999560534954071,
0.00024257962650153786,
0.0001967996940948069
] |
[
0.998855471611023,
0.0007001254707574844,
0.00037331419298425317,
0.00007115455809980631
] |
en
| 0.999994 |
Mice were perfused before harvesting the organs according to a published protocol ( 41 ). Briefly, mice were perfused with PBS containing 75 U/ml heparin (Sigma-Aldrich). Lung tissue was minced and incubated with constant stirring for 30 min at 37°C in HBSS containing 1.3 mM EDTA (Sigma-Aldrich). The lung tissue was next treated with 150 U/ml collagenase (Sigma-Aldrich) in RPMI containing 5% FCS for 1 h at 37°C. The resulting suspension was pelleted, resuspended in 44% Percoll (Amersham Biosciences), layered on 67.5% Percoll, and centrifuged at 600 g at 4°C for 15 min. Liver tissue was homogenized, pelleted, resuspended in 35% Percoll containing 200 U/ml heparin, layered on 67.5% Percoll, and centrifuged at 600 g at 4°C for 15 min. Lung and liver lymphocytes at the gradient interface were harvested and washed twice before use.
|
14623912_p13
|
14623912
|
Harvest of Peripheral Organs.
| 4.142365 |
biomedical
|
Study
|
[
0.9994707703590393,
0.00034982801298610866,
0.00017939160170499235
] |
[
0.9956058859825134,
0.003621605457738042,
0.0006056478596292436,
0.00016678698011673987
] |
en
| 0.999997 |
For the naive OT-I T cell population used for real-time RT-PCR, splenocytes from naive OT-I mice were purified on a nylon wool column to enrich for T cells and then stained for CD8, Vα2, and CD44. After washing, the splenocyte population was sorted on the basis of CD8 + , Vα2 + , and CD44 lo using a MoFlo™ high speed sorter (Cytomation, Inc.). The effector OT-I T cell population was obtained from B6.Thy1.1 mice that were transferred with OT-I T cells and then primed with LM/OVA. 7 d postinfection, the mice were killed and the splenocytes were purified on a nylon wool column. The resulting cells were stained for CD8 and Thy1.2 and sorted for expression of these molecules. For the memory OT-I T cell population, the same protocol was used as for the effector population, except the mice were killed at >4 wk postinfection. For each of the sorted OT-I T cell populations, the purity of the cells was >95% as determined by flow cytometry after sorting.
|
14623912_p14
|
14623912
|
Real-time RT-PCR.
| 4.168265 |
biomedical
|
Study
|
[
0.9995222091674805,
0.00027102642343379557,
0.00020671724632848054
] |
[
0.9989842772483826,
0.0005932311178185046,
0.0003517962177284062,
0.00007065937825245783
] |
en
| 0.999998 |
RNA was purified from the sorted naive, effector, and memory OT-I T cell populations using QIAGEN RNeasy columns. The protocol provided by the manufacturer was followed with the addition of the on column DNA digestion using the RNase-free DNase set from QIAGEN in order to remove any residual, contaminating DNA. cDNA was then synthesized from the RNA using the oligo-dT method and TaqMan™ reverse transcription reagents from Applied Biosystems.
|
14623912_p15
|
14623912
|
Real-time RT-PCR.
| 4.125284 |
biomedical
|
Study
|
[
0.9995744824409485,
0.00023898115614429116,
0.00018644664669409394
] |
[
0.9881445169448853,
0.010659871622920036,
0.0009637612965889275,
0.00023178815899882466
] |
en
| 0.999998 |
Real-time RT-PCR was performed on each of the three cDNA populations to amplify GAPDH (as a control to normalize for the quantity of cDNA), IL-12Rβ1, IL-12Rβ2, IL-18Rα, and IL-18Rβ. The primers used are listed from 5′ to 3′: GAPDH sense, TGCACCACCAACTGCTTAG and GAPDH antisense, GGATGCAGGGATGATGTTC; IL-12Rβ1 sense, GGCAACATGACATCCATGCA and IL-12Rβ1 antisense, GTGTGTCACCATCTTGGCAGGATC; IL-12Rβ2 sense, CACTATCAGGTGACGTTACA and IL-12Rβ2 antisense, TGCAG-AAGCGCCTTTTGAGTTGGT; IL-18Rα sense, GTGCACAGGAATGAAACAGC and IL-18Rα antisense, ATTTAAGGTCCAATTGCGACGA; and IL-18Rβ sense, GGAGTGGGAAATGTCAGTAT and IL-18Rβ antisense, CCGTGCC-GAGAAGGATGTAT. An Applied Biosystems GeneAmp 5700 thermocycler was used in conjunction with the SYBR Green™ PCR Master Mix, also from Applied Biosystems. Each sample was run in triplicate, and the data were analyzed using the 2 -ΔΔC T method ( 42 ) in order to determine the relative gene expression of each of the IL-12 and IL-18 receptors.
|
14623912_p16
|
14623912
|
Real-time RT-PCR.
| 4.133577 |
biomedical
|
Study
|
[
0.999574601650238,
0.00022681547852698714,
0.0001986280403798446
] |
[
0.9989929795265198,
0.0006106249638833106,
0.00032778215245343745,
0.00006865061004646122
] |
en
| 0.999998 |
Fig. S1 shows the ability of effector P14 TCR transgenic T cells generated in vivo to respond in vitro to either a combination of IL-12 and IL-18 or their cognate peptide. After overnight culture of splenocytes containing effector P14 T cells with the stimuli shown, the T cells secrete IFN-γ. This indicates that in addition to OT-I T cells, transgenic CD8 + T cells with another specificity are responsive to IL-12 and IL-18. Fig. S2 depicts the response of memory OT-I T cells generated in vivo to both LPS and Escherichia coli . After culturing splenocytes containing memory OT-I T cells in vitro for 3 h in the presence of GolgiPlug and the indicated stimuli, the OT-I T cells secrete IFN-γ. This result indicates that memory T cells have the ability to secrete IFN-γ in response to pathogens other than LM. Fig. S3 compares the ability of sorted naive or memory OT-I T cells to provide protection from LM infection in IFN-γ 2/− mice. The memory OT-I T cells provide protection from a LM infection as shown by the lowered LM colony counts in both the spleen and liver. The naive OT-I T cells do not provide protection from LM in either the spleen or the liver when they are transferred into IFN-γ 2/− mice. Figs. S1–S3 are available at http://www.jem.org/cgi/content/full/jem.20031051/DC1 .
|
14623912_p17
|
14623912
|
Online Supplemental Material.
| 4.171566 |
biomedical
|
Study
|
[
0.9996110796928406,
0.00021982098405715078,
0.00016913385479710996
] |
[
0.9990167617797852,
0.0004409962275531143,
0.00046864987234584987,
0.00007364523480646312
] |
en
| 0.999997 |
We demonstrated previously that a population of CD8 + T cells isolated from naive mice had the ability to respond to LM-infected macrophages by secreting IFN-γ ( 36 ). To further investigate the potential of CD8 + T cells to respond in vivo to LM, we used an adoptive transfer protocol where T cell–enriched splenocytes isolated from OT-I TCR transgenic mice were transferred into B6.Thy1.1 congenic hosts. The mice were then challenged with a strain of LM expressing the ovalbumin protein (LM/OVA) to induce the activation of the OT-I T cells. 7 d after infection, the CD8 + , Thy1.2 + population identifies the transferred OT-I T cells, which express both Vα2 and Vβ5 and also stain positive using a K b -SIINFEKL tetramer . The OT-I population of T cells express the CD44 and CD94 molecules indicative of an activated phenotype , and splenocytes from these mice are cytolytic against targets pulsed with the SIINFEKL peptide (unpublished data). When splenocytes from these OT-I–transferred, LM/OVA-primed mice were cultured overnight with SIINFEKL or a combination of IL-12 and IL-18, intracellular IFN-γ is detected in the majority of the CD8 + , Thy1.2 + T cells . In addition, stimulation of the CD8 + , Thy1.2 + T cells to secrete IFN-γ with LM/OVA-infected J774:K b macrophages could not be blocked by antibodies against IL-12 and IL-18. In contrast, IFN-γ secretion induced by LM/OVA-infected J774, LM-infected J774, or LM-infected J774:K b was blocked by antibodies against IL-12 and IL-18.
|
14623912_p18
|
14623912
|
Responses of Effector CD8 + T Cells to IL-12 and IL-18.
| 4.347003 |
biomedical
|
Study
|
[
0.9994063377380371,
0.00037708025774918497,
0.00021657883189618587
] |
[
0.9991742968559265,
0.00033011214691214263,
0.0003844538878183812,
0.00011104715667897835
] |
en
| 0.999997 |
To examine the effect of priming OT-I T cells with other pathogens, we infected B6.Thy1.1 mice that had been transferred with T cell–enriched OT-I splenocytes with VV-expressing ovalbumin (VV/OVA) or VSV-expressing ovalbumin (VSV/OVA). 7 d after infection, we analyzed the cell surface phenotype of the CD8 + , Thy1.2 + T cells to confirm that they had responded to antigen . After overnight stimulation, the OT-I T cells primed with either VV/OVA or VSV/OVA are able to respond to IL-12 and IL-18 by secreting IFN-γ . These effector T cells respond to LM-infected J774:K b by secreting IFN-γ even though the cognate antigen (OVA) is not provided. Antibodies against IL-12 and IL-18 block the IFN-γ secretion induced by LM-infected J774:K b but not LM/OVA-infected J774:K b . Similar responsiveness to IL-12 and IL-18 was observed using P14 TCR transgenic CD8 + T cells specific for lymphocytic choriomeningitis virus–derived glycoprotein 33–41 as an effector population generated in vivo .
|
14623912_p19
|
14623912
|
Responses of Effector CD8 + T Cells to IL-12 and IL-18.
| 4.248896 |
biomedical
|
Study
|
[
0.9994248151779175,
0.00034451045212335885,
0.00023070804309099913
] |
[
0.999291181564331,
0.00029070451273582876,
0.00033815449569374323,
0.00007993177132448182
] |
en
| 0.999995 |
To further our understanding of the ability of CD8 + T cells to respond to IL-12 and IL-18, we used the same OT-I transfer protocol as above but allowed the mice to rest for >4 wk before analysis. During this time period, the OT-I T cells respond to the antigen and then return to a resting but memory state. B6.Thy1.1 mice transferred with OT-I T cells that were challenged with LM/OVA , VV/OVA , or VSV/OVA were analyzed for expression of memory and activation markers. The expression of CD44 and CD94 on each of the transferred populations indicates that the OT-I T cells have a memory phenotype. In addition, none of the memory OT-I T cell populations expressed the activation markers CD25 or CD69 (not depicted). After overnight culture of splenocytes obtained from these animals, the OT-I T cells responded equally well to IL-12 and IL-18 or the cognate peptide in terms of their IFN-γ response. Importantly, regardless of the OVA-expressing pathogen used to generate memory OT-I T cells, LM-infected J774:K b stimulators induced IFN-γ secretion, and this effect was blocked by antibodies against IL-12 and IL-18 .
|
14623912_p20
|
14623912
|
Responses of Memory CD8 + T Cells to IL-12 and IL-18.
| 4.236927 |
biomedical
|
Study
|
[
0.9994571805000305,
0.00033278498449362814,
0.00021003461733926088
] |
[
0.9993083477020264,
0.0002522035501897335,
0.00035764940548688173,
0.0000817987383925356
] |
en
| 0.999996 |
Our previous work has shown that a very small percentage of OT-I T cells stimulated directly ex vivo from an OT-I mouse was able to secrete IFN-γ ( 36 ). To analyze a naive population of transferred CD8 + T cells, T cell–enriched OT-I splenocytes were injected into B6.Thy1.1 hosts, and their potential to secrete IFN-γ without challenging the mice in vivo was determined. Indeed, a small percentage of the transferred OT-I T cells was able to secrete IFN-γ in response to either IL-12 and IL-18 or SIINFEKL . The IFN-γ–secreting OT-I T cells altered their cell surface phenotype compared with nontransferred OT-I T cells with respect to CD44, CD94, and other memory markers (unpublished data). These changes may explain why some transferred OT-I T cells are slightly more responsive to IL-12 and IL-18 compared with the nontransferred population of OT-I T cells. However, the nonprimed, transferred OT-I T cell population contained a much lower percentage of IL-12– and IL-18–responsive T cells compared with a memory population of OT-I T cells. Furthermore, when we transferred OT-I T cells into B6.Thy1.1 hosts and then attempted to prime with WT LM there was no proliferation of the OT-I T cells as measured by CFSE labeling experiments (unpublished data). Together, these data indicate that only effector and memory CD8 + T cells have the capacity to secrete IFN-γ in response to IL-12 and IL-18 and that this response is not mediated through the TCR.
|
14623912_p21
|
14623912
|
Responses of Memory CD8 + T Cells to IL-12 and IL-18.
| 4.310347 |
biomedical
|
Study
|
[
0.9993896484375,
0.00034995979513041675,
0.00026046985294669867
] |
[
0.9992334842681885,
0.0003528479137457907,
0.00033187828375957906,
0.00008178169809980318
] |
en
| 0.999997 |
To analyze a population of antigen-specific, non-TCR transgenic CD8 + T cells, we injected B6 mice with LM/OVA and identified endogenous OVA-specific CD8 + T cells using K b -SIINFEKL tetramers . Once again, both effector (7 d postinfection) and memory (>4 wk postinfection) OVA-specific CD8 + T cells secreted IFN-γ when stimulated overnight with a combination of IL-12 and IL-18 or their cognate peptide . This result, along with our previous work using bulk populations of CD8 + T cells, ( 36 ) suggests that multiple populations of effector and memory CD8 + T cells respond to IL-12 and IL-18 by secreting IFN-γ.
|
14623912_p22
|
14623912
|
Responses of Memory CD8 + T Cells to IL-12 and IL-18.
| 4.143873 |
biomedical
|
Study
|
[
0.9995201826095581,
0.0002524184819776565,
0.00022750456992071122
] |
[
0.9994434714317322,
0.0002420553209958598,
0.0002546862524468452,
0.00005968773984932341
] |
en
| 0.999996 |
Previous reports indicate that memory T cells preferentially relocate in peripheral organs ( 41 , 43 ). When we compared the localization of naive (transferred, unprimed) OT-I T cells and memory (transferred, LM/OVA-primed) OT-I T cells, we found that the memory OT-I T cells preferentially relocated to nonlymphoid organs, whereas the naive OT-I T cells did not . Importantly, when we restimulated lymphocytes isolated from LNs, spleen, liver, and lung we found that memory OT-I T cells were able to respond to both SIINFEKL or a combination of IL-12 and IL-18 by secreting IFN-γ . When this experiment was repeated, we used SIINFEKL-pulsed J774:K b as stimulators to compensate for the low numbers of cells isolated from the lung, and this increased the percentage of IFN-γ–secreting OT-I T cells to ∼75% (unpublished data). These results indicate that not only do memory CD8 + T cells preferentially reside in peripheral organs but also that these T cells are capable of mounting an early, rapid response against a pathogen-induced infection.
|
14623912_p23
|
14623912
|
Localization of Memory CD8 + T Cells in Peripheral Organs and Their Ability To Respond To IL-12 and IL-18.
| 4.282234 |
biomedical
|
Study
|
[
0.9994843006134033,
0.0003319967654533684,
0.00018369729514233768
] |
[
0.9990907907485962,
0.00031732660136185586,
0.0005005775601603091,
0.00009131182014243677
] |
en
| 0.999997 |
To test whether or not memory OT-I T cells respond to a WT LM infection in vivo, we rechallenged OT-I–transferred, VSV/OVA-primed memory mice for 16 h with differing doses of WT LM. The splenocytes were then cultured for 3 h in media with GolgiPlug™ and subsequently analyzed for IFN-γ production. Indeed, memory OT-I T cells responded to WT LM in a dose-dependent fashion by secreting IFN-γ . The probable reason for the requirement of high doses of WT LM to induce a substantial percentage of the OT-I T cells to secrete IFN-γ is that the assay was performed after only 16 h of infection. In other experiments, ∼50% of the memory OT-I T cells were able to secrete IFN-γ 3 d postinfection with the sublethal dose of ∼10 4 WT LM (unpublished data). OT-I–transferred, VSV/OVA-primed memory mice were also injected with IL-12 and IL-18. Once again, ∼50% of the OT-I T cells stained positive for intracellular IFN-γ (unpublished data). These results suggest that the combined actions of IL-12 and IL-18 are responsible for inducing rapid IFN-γ secretion in memory CD8 + T cells responding to WT LM. To expand these findings to another organism, we infected memory OT-I mice with E. coli and killed the mice 3 h later. ∼40% of the memory OT-I T cells isolated from the E. coli –infected mice secreted IFN-γ . Together, these results indicate that memory CD8 + T cells have the ability to respond to multiple pathogens in an antigen-independent fashion.
|
14623912_p24
|
14623912
|
Rapid In Vivo Production of IFN-γ from Memory CD8 + T Cells in Response To WT LM.
| 4.360986 |
biomedical
|
Study
|
[
0.9993852376937866,
0.000407513725804165,
0.00020717813458759338
] |
[
0.9989466071128845,
0.0004427194071467966,
0.0005000282544642687,
0.00011067013838328421
] |
en
| 0.999996 |
To establish a mechanism for the increased IFN-γ responsiveness of effector and memory OT-I T cells to a combination of IL-12 and IL-18 compared with their naive counterparts, we performed real-time RT-PCR on sorted populations of naive, effector, and memory OT-I T cells. Levels of IL-12Rβ2, IL-18Rα, and IL-18Rβ were all up-regulated in the effector and memory OT-I T cell populations . To confirm these findings at the protein level, we analyzed naive, effector, and memory populations of OT-I T cells for expression of the IL-18Rα subunit. Although in each population of OT-I T cells the IL-18Rα subunit is expressed, the effector and memory populations express approximately a fivefold higher level than the naive population . The flow cytometry data, which supports the RT-PCR data for the IL-18Rα subunit, suggests that the antibodies used for sorting to obtain the pure populations of OT-I T cells did not affect the gene expression of the IL-12 and IL-18 receptors. In addition, we observed that memory OT-I T cells generated by priming with either VV/OVA or VSV/OVA expressed high levels of the IL-18Rα subunit, explaining their increased responsiveness to IL-12 and IL-18 (unpublished data).
|
14623912_p25
|
14623912
|
Up-regulation of IL-12 and IL-18 Receptors in Effector and Memory CD8 + T Cells.
| 4.246732 |
biomedical
|
Study
|
[
0.9994671940803528,
0.00031227825093083084,
0.0002205723721999675
] |
[
0.999377965927124,
0.0002561476139817387,
0.00029178051045164466,
0.00007408226520055905
] |
en
| 0.999998 |
To investigate the early kinetics of the innate requirements for IFN-γ during an LM infection, we compared the susceptibility of B6 and IFN-γ −/− mice infected with ∼10 4 LM on days 1, 2, and 3 postinfection. The differences in LM susceptibility, as determined by LM counts in the spleen and liver, are only detectable between 2–3 d postinfection . These data indicate that IFN-γ is important during the innate immune response to LM, but the importance of this effector cytokine does not manifest itself immediately after infection.
|
14623912_p26
|
14623912
|
Differences in LM Susceptibility Between B6 and IFN-γ −/− Mice.
| 4.15356 |
biomedical
|
Study
|
[
0.9994950294494629,
0.00031619565561413765,
0.00018868682673200965
] |
[
0.9992135763168335,
0.00027848652098327875,
0.00043818264384754,
0.00006966420187382028
] |
en
| 0.999996 |
Our data suggest that antigen nonspecific secretion of IFN-γ in response to WT LM by memory CD8 + T cells may play a role in protective immunity against LM and that this response can be visualized within 16 h . Furthermore, differences in LM counts mediated by IFN-γ can only be seen 2–3 d postinfection . Therefore, we transferred memory OT-I T cells into IFN-γ −/− mice infected with ∼10 4 LM and determined spleen and liver LM counts 3 d postinfection. Spleen and liver LM counts for IFN-γ −/− mice on day 3 after infection show high numbers of bacterial colonies compared with WT B6 control animals, as expected . In contrast, IFN-γ −/− mice that received 5 × 10 5 sorted memory OT-I T cells before LM infection show ∼2 logs of protection in both organs. We were able to detect CFSE staining of the transferred memory OT-I T cells 3 d postinfection . As few as ∼5,000 IFN-γ–secreting OT-I T cells are able to reduce the LM counts in IFN-γ −/− mice almost to the levels of a B6 mouse. The small number of OT-I T cells recovered 3 d postinfection is most likely attributed to loss upon injection, localization to other organs, and the fact that not all of the OT-I T cells secrete IFN-γ at the time of the assay. Importantly, there are ∼290-fold fewer IFN-γ–secreting cells in the IFN-γ −/− mice transferred with memory OT-I T cells compared with the B6 mice . It should be noted that this response does not represent cross-reactivity between the SIINFEKL epitope and LM since the transferred cells show no evidence of dilution of the CFSE label . Our previous data indicate that naive OT-I T cells are not responsive to IL-12 and IL-18 and that they do not express high levels of the receptors for these cytokines . In accordance with this data, when we transferred naive OT-I T cells into IFN-γ −/− mice they were unable to protect these animals from a WT LM infection . These results are also demonstrated in an independent experiment where we transferred naive or memory OT-I T cells into IFN-γ–deficient mice and challenged both sets of mice with 10 4 WT LM .
|
14623912_p27
|
14623912
|
Early Protection of IFN-γ −/− Mice from LM Infection by Memory CD8 + T Cells Independent of Antigen Specificity.
| 4.36586 |
biomedical
|
Study
|
[
0.9993072748184204,
0.00047715535038150847,
0.00021567029762081802
] |
[
0.9991214871406555,
0.00031726222368888557,
0.00043047021608799696,
0.00013079169730190188
] |
en
| 0.999998 |
CD8 + T cells play an important role in the control and elimination of LM and other intracellular pathogens. Signals propagated through the TCR upon binding to peptide–MHC complexes can induce a myriad of effects including proliferation, cytokine secretion, cytolysis of infected targets, and apoptosis of the T cells themselves ( 44 ). Generation of memory CD8 + T cells results in rapid responses upon reexposure to the same pathogen. In addition, memory CD8 + T cells have been shown to preferentially localize to peripheral nonlymphoid organs ( 41 , 43 ). Recent reports in both human and murine systems have suggested that memory CD8 + T cells may belong to distinct subsets, termed central and effector, based upon their existence in either lymphoid or nonlymphoid organs ( 45 , 46 ). Central memory CD8 + T cells are CD62L hi and CCR7 + allowing homing to LNs, whereas effector memory CD8 + T cells are CD62L lo and CCR7 − and do not have this homing property. Effector memory CD8 + T cells have been proposed to respond rapidly to a secondary infection by performing effector functions ( 46 ), although this has been questioned recently ( 45 ). Data presented here indicate that memory CD8 + T cells can respond rapidly in an antigen nonspecific manner by secreting IFN-γ . We have isolated these memory CD8 + T cells from both lymphoid and nonlymphoid organs . Therefore, they may belong to either subset of memory CD8 + T cells. Furthermore, we have shown previously that both CD62L hi and CD62L lo CD8 + T cells have the capacity to secrete IFN-γ upon exposure to IL-12 and IL-18 ( 36 ). Importantly, the localization of memory CD8 + T cells to sites of infection such as the GI tract, lung, and liver make our findings relevant in terms of an immediate response against an infection.
|
14623912_p28
|
14623912
|
Discussion
| 4.524014 |
biomedical
|
Study
|
[
0.9992052912712097,
0.0005160091095604002,
0.0002787839330267161
] |
[
0.9980704188346863,
0.0004577901854645461,
0.0012919992441311479,
0.0001797995500965044
] |
en
| 0.999997 |
IFN-γ is critical in immune responses against certain pathogens. For example, the published LD 50 of IFN-γ–deficient Balb/c mice in response to LM is ∼10 ( 31 ). However, LM-specific CD8 + T cells generated in these mice can provide protective immunity to future LM infections. In addition, studies using SCID mice lacking T and B lymphocytes have demonstrated increased LM burdens upon depletion of IFN-γ ( 33 ). These results suggest that IFN-γ is required during the innate immune response to LM. Indeed, our data indicate that IFN-γ–deficient mice are highly susceptible to LM infections but that the differences in LM burden between B6 and IFN-γ −/− mice are not apparent until 2–3 d postinfection . Although IFN-γ can have direct effects on LM growth by limiting escape from the phagosome ( 47 ), the major role of this cytokine may be in inducing macrophage activity or polarizing toward a Th1-type immune response ( 2 ). Exactly what role the IFN-γ that is secreted rapidly from antigen nonspecific memory CD8 + T cells plays was not determined in our studies. However, it is involved in controlling the early, rapid growth of LM that would otherwise occur in IFN-γ–deficient mice .
|
14623912_p29
|
14623912
|
Discussion
| 4.312151 |
biomedical
|
Study
|
[
0.9995378255844116,
0.0002673562557902187,
0.0001948619174072519
] |
[
0.9991402626037598,
0.0003133527934551239,
0.00045778515050187707,
0.0000886634734342806
] |
en
| 0.999996 |
Our experiments have used three different pathogens, all expressing the OVA protein, to induce effector and memory CD8 + T cells. In each case, the CD8 + T cells acquired the ability to secrete IFN-γ in response to IL-12 and IL-18 and up-regulated the expression of the IL-12 and IL-18 receptors . Whether or not IL-12 and IL-18 production during the priming phase of CD8 + T cell activation is required for the increase in expression of the receptors for the cytokines is unclear. Signaling through the TCR upon binding to the cognate peptide–MHC complex may result in up-regulation of the IL-12 and IL-18 receptors in CD8 + T cells. It is likely that for each of the pathogens used the OVA antigen (SIINFEKL) is actually presented by DC through cross-priming ( 48 ). Recent work suggests that CD8 + DC are responsible for cross-priming CD8 + T cells and that this subset of DC produce high amounts of IL-12 ( 49 , 50 ). DC of different types are also capable of secreting IL-18 ( 51 ). Therefore, it seems likely that IL-12 and IL-18 may be produced during the priming phase required to induce responsiveness of the CD8 + T cells to IL-12 and IL-18.
|
14623912_p30
|
14623912
|
Discussion
| 4.288321 |
biomedical
|
Study
|
[
0.9994282126426697,
0.0003194055170752108,
0.0002523103030398488
] |
[
0.9992499947547913,
0.00025227974401786923,
0.00042153926915489137,
0.00007612571062054485
] |
en
| 0.999999 |
It is interesting to note that IL-12 and IL-18 did not induce any other effector functions, including proliferation, cytotoxicity, or secretion of TNF-α from effector or memory CD8 + T cells (unpublished data). Recent studies have demonstrated that IL-12 can have other effects on T cells, such as inducing the indirect proliferation of memory CD8 + T cells ( 52 ). IL-12 has also been shown to be necessary for complete activation of naive CD8 + T cells but not memory CD8 + T cells ( 53 ). IL-18 has been implicated in up-regulating the cytotoxic activities of CD8 + T cells ( 23 ). Our results indicate that the secretion of cytokine-induced IFN-γ by CD8 + T cells is under exquisite control. Once IL-12 and IL-18 are no longer produced, which should be rapid under conditions of a controllable infection, then IFN-γ will cease to be secreted from effector and memory CD8 + T cells.
|
14623912_p31
|
14623912
|
Discussion
| 4.183475 |
biomedical
|
Study
|
[
0.9994812607765198,
0.0002756226167548448,
0.00024321427918039262
] |
[
0.9994144439697266,
0.00025891707628034055,
0.00026804034132510424,
0.000058557652664603665
] |
en
| 0.999999 |
The most striking result of this study is that so few memory CD8 + T cells can protect IFN-γ–deficient mice 3 d post LM infection . Considering other cell types can respond rapidly to LM by secreting IFN-γ, there may be redundancy in this early response. Alternatively, the extra IFN-γ production resulting from multiple cell types may be required in order to establish a Th1 environment, aiding in the generation and effectiveness of the adaptive immune response. The ability of NK, NK-T, and γδ T cells to provide innate immune protection is a possibility that is being considered. In conclusion, the results presented here demonstrate that IFN-γ production from antigen nonspecific memory CD8 + T cells is sufficient in providing innate immune protection against LM. Furthermore, this study highlights the fact that CD8 + T cells can function in settings other than the adaptive immune response.
|
14623912_p32
|
14623912
|
Discussion
| 4.170161 |
biomedical
|
Study
|
[
0.9995224475860596,
0.00030178355518728495,
0.0001757017453201115
] |
[
0.9990559220314026,
0.00023644728935323656,
0.0006321104592643678,
0.000075497679063119
] |
en
| 0.999997 |
Replication protein A (RPA) was identified as a heterotrimeric single-stranded DNA (ssDNA)-binding protein required for replication of simian virus 40 (SV40) DNA in vitro [for reviews see ( 1 – 6 )]. RPA is now known to be essential for chromosomal DNA replication, repair and recombination pathways in eukaryotic cells, and new roles in DNA damage signaling and regulation of replication origin firing frequency are emerging ( 7 – 15 ). RPA functions to protect ssDNA from nucleases and prevent hairpin formation in ssDNA that would interfere with DNA processing, but it also appears to actively coordinate the sequential assembly and disassembly of DNA processing proteins on ssDNA ( 16 , 17 ). The ability of RPA to guide DNA processing depends in large part on RPA interactions with other proteins in each pathway. Although these mechanisms are not yet well understood, we will review here several examples and discuss possible models for protein-mediated RPA conformation changes that may underlie its assembly and disassembly on ssDNA.
|
16935876_p0
|
16935876
|
INTRODUCTION
| 4.391107 |
biomedical
|
Review
|
[
0.9908808469772339,
0.004881555680185556,
0.004237594548612833
] |
[
0.015472173690795898,
0.0013028738321736455,
0.9825980067253113,
0.0006268809665925801
] |
en
| 0.999997 |
RPA is a stable complex of three subunits RPA70, RPA32 and RPA14 that are conserved among eukaryotes . The 3D structures of RPA fragments reveal six domains that adopt an oligonucleotide binding (OB)-fold, a structure common to other known SSBs ( 13 , 18 ) . Zinc binding in the C-terminal OB-fold of RPA70 is important for RPA structural stability and ssDNA binding ( 19 ). RPA32C adopts a winged-helix–turn–helix fold ( 20 ). Flexible linkers join the domains in each subunit and the two small subunits interact with RPA70C through a 3-helix bundle. However, the quaternary structure(s) of RPA remains elusive.
|
16935876_p1
|
16935876
|
RPA: A MODULAR PROTEIN WITH MULTIPLE CONFORMATIONS
| 4.456936 |
biomedical
|
Study
|
[
0.9994993209838867,
0.0002453566703479737,
0.00025530214770697057
] |
[
0.9965817332267761,
0.0006695401971228421,
0.00263423309661448,
0.00011449512385297567
] |
en
| 0.999995 |
RPA binds tightly to ssDNA with a defined 5′→3′ polarity ( 21 , 22 ), and an affinity of up to ∼10 −9 –10 −10 M ( 23 ). RPA contains four ssDNA-binding domains (A–D in order of decreasing affinities), three in the RPA70 subunit, tethered to each other through flexible linkers, and one in the RPA32 subunit. RPA binds to ssDNA in at least three different modes characterized by the length of ssDNA that it contacts (8–10, 12–23 and 28–30 nt) and the number of ssDNA-binding domains involved ( 24 – 26 ). In the 30 nt binding mode, the 5′ end of the binding site is occupied by RPA70A and the 3′ end by RPA32D ( 21 , 22 , 24 , 27 – 29 ). The N-terminal OB-fold of RPA70 (RPA70N) also has weak (mM) ssDNA-binding affinity and may contribute to regulation of ssDNA-binding mode under some conditions ( 30 – 32 ). The three ssDNA-binding modes of heterotrimeric RPA imply that the protein can adopt three different structural conformations. Indeed scanning transmission electron micrographs and gel filtration demonstrate RPA molecules in compact and extended conformations on ssDNA ( 33 ). These studies suggest that all three ssDNA-binding modes co-exist in solution, perhaps in equilibrium ( 33 ), but the intramolecular structural re-organization of RPA domains that gives rise to the three binding modes remains unknown.
|
16935876_p2
|
16935876
|
RPA: A MODULAR PROTEIN WITH MULTIPLE CONFORMATIONS
| 4.622591 |
biomedical
|
Study
|
[
0.9991567134857178,
0.0004797420115210116,
0.0003635339962784201
] |
[
0.9954458475112915,
0.0005517181707546115,
0.0038085931446403265,
0.0001938093191711232
] |
en
| 0.999997 |
A great deal of insight into RPA interactions with ssDNA has been obtained from crystal structures of RPA70AB determined in the presence and absence of dC 8 ( 25 , 34 ) . In the DNA-free state, two different relative orientations of the 70A and 70B domains were found, one of them with an unstructured linker between OB-fold A and B ( 25 ), suggesting that the linker between the A and B domains is flexible. This flexibility has been independently confirmed by NMR studies of RPA70AB in solution ( 29 ). In the presence of ssDNA, OB-fold domains A and B align in a fixed orientation with the linker parallel to the bound oligonucleotide, but on the opposite side of the protein ( 34 ). Binding of RPA70AB to ssDNA is accompanied by conformational changes, in which two extended loops in each domain close like ‘fingers’ around the DNA. The structural re-organization within each domain and the change in dynamics between the domains imply that RPA must pay a significant entropic penalty to bind ssDNA.
|
16935876_p3
|
16935876
|
RPA: A MODULAR PROTEIN WITH MULTIPLE CONFORMATIONS
| 4.511119 |
biomedical
|
Study
|
[
0.9992994070053101,
0.00041421939386054873,
0.00028635578928515315
] |
[
0.9977164268493652,
0.00039073015796020627,
0.0017523964634165168,
0.00014045333955436945
] |
en
| 0.999997 |
Binding to ssDNA in the 30 nt binding mode is thought to progress sequentially from 5′ to 3′, beginning with the RPA70 domains A and B in an initial 10 nt binding mode . The ssDNA-binding affinity of these individual DNA-binding domains is quite weak. RPA70A binds to ssDNA with greater affinity than the other ssDNA-binding domains ( K d ∼ 2 μM), but because only a short linker separates RPA70A and RPA70B, the local concentration of 70B is high when 70A binds to ssDNA ( 29 ). This leads to ssDNA binding of both domains. In the heterotrimeric RPA molecule, this chemical linkage between the weak individual binding domains enhances the overall affinity of RPA for an ssDNA molecule by several orders of magnitude ( 29 ), progressing to the high-affinity-binding mode that involves all four major ssDNA-binding domains ( 21 , 22 , 24 , 25 , 28 , 29 ) . Thus RPA ‘unrolls’ readily on ssDNA, forming a stable complex that occludes ∼30 nt, stabilizes the ssDNA against nuclease digestion and stabilizes the protein against proteolytic digestion.
|
16935876_p4
|
16935876
|
RPA: A MODULAR PROTEIN WITH MULTIPLE CONFORMATIONS
| 4.698982 |
biomedical
|
Study
|
[
0.9990729093551636,
0.0006086518988013268,
0.00031845507328398526
] |
[
0.9947320222854614,
0.0017527012387290597,
0.0032003873493522406,
0.00031484224018640816
] |
en
| 0.999996 |
DNA processing involves not only RPA binding to ssDNA substrates as discussed above, but also to partial duplexes with a variety of structures. Photoaffinity labeling studies with partial duplex DNA uncovered a novel binding mode in which RPA32D and 70C bind to the 3′-OH of a partial duplex and to a 5′-single-stranded overhanging end ( 35 – 37 ) . Labeling of RPA32D predominated using 3′OH photoaffinity labels with short crosslinker spacers, while RPA70C was labeled using longer spacers, indicating the relative proximity of the two domains to the 3′OH at the primer–template junction. Strikingly, the RPA trimerization core (RPA70C-32D-14) alone was sufficient for this binding mode on partial duplex with a 5′ ssDNA overhang of either 10 or 30 nt ( 24 , 38 ), implying that intact RPA may also use this binding mode to bind to primer–template junctions or DNA with single-stranded gaps of <30 nt. The observation that the junction-binding mode does not utilize RPA70AB raises the question of whether an RPA molecule in the 8–10 nt ssDNA-binding mode may actually have two faces able to bind different DNA structures, one composed of RPA70AB and another composed of the trimerization core. Elucidation of RPA quaternary structures will be needed to answer the question.
|
16935876_p5
|
16935876
|
RPA: A MODULAR PROTEIN WITH MULTIPLE CONFORMATIONS
| 4.482149 |
biomedical
|
Study
|
[
0.9993919134140015,
0.00040551519487053156,
0.00020255730487406254
] |
[
0.998248815536499,
0.0005210107192397118,
0.0010737119009718299,
0.00015646965766791254
] |
en
| 0.999998 |
RPA tightly bound to ssDNA during DNA processing must somehow be displaced to allow completion of the processing pathway and restoration of the base-paired DNA. How RPA dissociation occurs is not well understood, but one possibility is that the four ssDNA-binding domains dissociate sequentially in the reverse order, i.e. from the 3′ to the 5′ end of the ssDNA . Given that RPA binds tightly to ssDNA, it seems likely that its complete dissociation from ssDNA requires the participation of other proteins in each DNA processing pathway. We suggest that these proteins bind to RPA, inducing a change in its conformation to a weaker binding mode and thereby facilitating its dissociation from ssDNA.
|
16935876_p6
|
16935876
|
RPA: A MODULAR PROTEIN WITH MULTIPLE CONFORMATIONS
| 4.444676 |
biomedical
|
Study
|
[
0.9993755221366882,
0.00036510758218355477,
0.0002593164099380374
] |
[
0.9959105253219604,
0.0029232085216790438,
0.0009438248816877604,
0.0002224181080237031
] |
en
| 0.999998 |
Subsets and Splits
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