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  • 1
    Online-Ressource
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    Elsevier BV ; 2002
    In:  Experimental Hematology Vol. 30, No. 10 ( 2002-10), p. 1147-1152
    In: Experimental Hematology, Elsevier BV, Vol. 30, No. 10 ( 2002-10), p. 1147-1152
    Materialart: Online-Ressource
    ISSN: 0301-472X
    RVK:
    Sprache: Englisch
    Verlag: Elsevier BV
    Publikationsdatum: 2002
    ZDB Id: 2005403-8
    Standort Signatur Einschränkungen Verfügbarkeit
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  • 2
    Online-Ressource
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    Wiley ; 2002
    In:  Cytometry Vol. 47, No. 2 ( 2002-02), p. 89-99
    In: Cytometry, Wiley, Vol. 47, No. 2 ( 2002-02), p. 89-99
    Kurzfassung: Telomeres containing noncoding DNA repeats at the end of the chromosomes are essential for chromosomal stability and are implicated in regulating the replication and senescence of cells. The gradual loss of telomere repeats in cells has been linked to aging and tumor development and methods to measure telomere length are of increasing interest. At least three methods for measuring the length of telomere repeats have been described: Southern blot analysis and quantitative fluorescence in situ hybridization using either digital fluorescence microscopy (Q‐FISH) or flow cytometry (flow‐FISH). Both Southern blot analysis and Q‐FISH have specific limitations and are time‐consuming, whereas the flow‐FISH technique requires relatively few cells (10 5 ) and can be completed in a single day. A further advantage of the flow‐FISH method is that data on the telomere length from individual cells and subsets of cells (lymphocytes and granulocytes) can be acquired from the same sample. In order to obtain accurate and reproducible results using the flow‐FISH technique, we systematically explored the influence of various steps in the protocol on telomere length values and established an acceptable range for the most critical parameters. Methods Isolated leukocytes from whole blood are denatured by heat and 70%/75% formamide, then hybridized with or without a telomere‐specific fluorescein isothiocyante (FITC)‐conjugated peptide nucleic acid probe (PNA). Unbound telomere PNA is washed away, the DNA is counterstained, and telomere fluorescence is measured on a flow cytometer using an argon ion laser (488 nm) to excite FITC. For each sample, duplicates of telomere PNA‐stained and unstained tubes are analyzed. Results Cell counts and flow‐FISH telomere length measurements were performed on leukocytes and thymocytes of humans and other species. Leukocyte suspensions were prepared by two red blood cell lysis steps with ammonium chloride. Optimal denaturation of DNA was achieved by heating at 85–87°C for 15 min in a solution containing 70%/75% formamide. Hybridization was performed at room temperature with a 0.3 μg/ml telomere‐PNA probe for at least 60–90 min. Unbound telomere‐PNA probe was diluted at least 4,000–40,000 times with wash steps containing 70%/75% formamide at room temperature. LDS 751 and DAPI were suitable as DNA counterstains as they did not show significant interference with telomere length measurement. Conclusions The use of flow‐FISH for telomere length measurements in nucleated blood cells requires tight adherence to an optimized protocol. The method described here can be used to determine rapidly the telomere length in subsets of nucleated blood cells. Cytometry 47:89–99, 2002. © 2002 Wiley‐Liss, Inc.
    Materialart: Online-Ressource
    ISSN: 0196-4763 , 1097-0320
    URL: Issue
    RVK:
    Sprache: Englisch
    Verlag: Wiley
    Publikationsdatum: 2002
    ZDB Id: 2180639-1
    ZDB Id: 1474272-X
    ZDB Id: 2180651-2
    SSG: 12
    Standort Signatur Einschränkungen Verfügbarkeit
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  • 3
    Online-Ressource
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    Wiley ; 2003
    In:  Cytometry Vol. 55A, No. 1 ( 2003-09), p. 1-6
    In: Cytometry, Wiley, Vol. 55A, No. 1 ( 2003-09), p. 1-6
    Materialart: Online-Ressource
    ISSN: 0196-4763 , 1097-0320
    URL: Issue
    RVK:
    Sprache: Englisch
    Verlag: Wiley
    Publikationsdatum: 2003
    ZDB Id: 2180639-1
    ZDB Id: 1474272-X
    ZDB Id: 2180651-2
    SSG: 12
    Standort Signatur Einschränkungen Verfügbarkeit
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  • 4
    In: The Lancet, Elsevier BV, Vol. 362, No. 9396 ( 2003-11), p. 1628-1630
    Materialart: Online-Ressource
    ISSN: 0140-6736
    RVK:
    Sprache: Englisch
    Verlag: Elsevier BV
    Publikationsdatum: 2003
    ZDB Id: 2067452-1
    ZDB Id: 3306-6
    ZDB Id: 1476593-7
    SSG: 5,21
    Standort Signatur Einschränkungen Verfügbarkeit
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  • 5
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    Rockefeller University Press ; 2004
    In:  The Journal of Experimental Medicine Vol. 200, No. 11 ( 2004-12-06), p. 1407-1417
    In: The Journal of Experimental Medicine, Rockefeller University Press, Vol. 200, No. 11 ( 2004-12-06), p. 1407-1417
    Kurzfassung: Human immunodeficiency virus (HIV)-specific CD8+ T cells persist in high frequencies in HIV-infected patients despite impaired CD4+ T helper response to the virus, but, unlike other differentiated effector cytotoxic T lymphocytes, most continue to express the tumor necrosis factor receptor family member CD27. Because the ligand for CD27 (CD70) is also overexpressed in HIV-infected hosts, we examined the nature of expression and potential functional consequences of CD27 expression on HIV-specific CD8+ T cells. Analysis of CD27+ and CD27− T cells derived from the same HIV-specific clone revealed that retention of CD27 did not interfere with acquisition of effector functions, and that after T cell receptor stimulation, CD27+ cells that concurrently were triggered via CD27 exhibited more resistance to apoptosis, interleukin 2 production, and proliferation than CD27− T cells. After transfer back into an HIV-infected patient, autologous HIV-specific CD27− T cells rapidly disappeared, but CD27+ T cells derived from the same clone persisted at high frequency. Our findings suggest that the CD27–CD70 interaction in HIV infection may provide CD27+ CD8+ T cells with a survival advantage and compensate for limiting or absent CD4+ T help to maintain the CD8 response.
    Materialart: Online-Ressource
    ISSN: 1540-9538 , 0022-1007
    RVK:
    Sprache: Englisch
    Verlag: Rockefeller University Press
    Publikationsdatum: 2004
    ZDB Id: 1477240-1
    Standort Signatur Einschränkungen Verfügbarkeit
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  • 6
    Online-Ressource
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    American Society of Hematology ; 2003
    In:  Blood Vol. 102, No. 3 ( 2003-08-01), p. 916-918
    In: Blood, American Society of Hematology, Vol. 102, No. 3 ( 2003-08-01), p. 916-918
    Kurzfassung: Mutations in the human telomerase RNA (TERC) occur in autosomal dominant dyskeratosis congenita (DKC). Because of the possibility that TERC mutations might underlie seemingly acquired forms of bone marrow failure, we examined blood samples from a large number of patients with aplastic anemia (AA), paroxysmal nocturnal hemoglobinuria (PNH), and myelodysplasia (MDS). Only 3 of 210 cases showed heterozygous TERC mutations: both nucleotide 305 (n305) (G & gt;A) and n322 (G & gt;A) were within the conserved region (CR) 4–CR5 domain; n450 (G & gt;A) was localized to the boxH/ACA domain. However, only one patient (with a mutation at n305 [G & gt;A]) had clinical characteristics suggesting DKC; her blood cells contained short telomeres and her sister also suffered from bone marrow failure. Another 21 patients with short telomeres did not show TERC mutations. Our results suggest that cryptic DKC, at least secondary to mutations in the TERC gene, is an improbable diagnosis in patients with otherwise typical AA, PNH, and MDS.
    Materialart: Online-Ressource
    ISSN: 0006-4971 , 1528-0020
    RVK:
    RVK:
    Sprache: Englisch
    Verlag: American Society of Hematology
    Publikationsdatum: 2003
    ZDB Id: 1468538-3
    ZDB Id: 80069-7
    Standort Signatur Einschränkungen Verfügbarkeit
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  • 7
    In: Blood, American Society of Hematology, Vol. 104, No. 11 ( 2004-11-16), p. 2832-2832
    Kurzfassung: Human telomerase uses a portion of its integral RNA component (hTER) as the template to synthesize telomeres at chromosome ends. hTER sequence polymorphisms have been observed in some patients with bone marrow failure syndromes such as aplastic anemia, but the functional significance of most such variants is unknown. Here, we report the functional characteristics of ten previously-described and two newly discovered hTER disease-associated polymorphisms. Most of these hTER variants adversely affected telomerase enzymatic function as measured in the telomerase reconstituted human cells. Similar loss-of-function effects were also seen directly in primary lymphocytes collected from two of the patients. The majority of the functional deficits stemmed from perturbations of the predicted hTER RNA secondary structure, and corresponded well with the degrees of telomere shortening observed in patients. In contrast, hTER variants anticipated to be inconsequential polymorphisms, which were also found in healthy subjects, did not interfere with telomerase function. Loss of telomerase activity and of telomere maintenance resulting from inherited hTER mutations may predispose some patients to aplastic anemia and other marrow failure disorders.
    Materialart: Online-Ressource
    ISSN: 0006-4971 , 1528-0020
    RVK:
    RVK:
    Sprache: Englisch
    Verlag: American Society of Hematology
    Publikationsdatum: 2004
    ZDB Id: 1468538-3
    ZDB Id: 80069-7
    Standort Signatur Einschränkungen Verfügbarkeit
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  • 8
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    Wiley ; 2003
    In:  Annals of the New York Academy of Sciences Vol. 996, No. 1 ( 2003-05), p. 44-48
    In: Annals of the New York Academy of Sciences, Wiley, Vol. 996, No. 1 ( 2003-05), p. 44-48
    Kurzfassung: A bstract : Hematopoietic stem cells have an impressive regenerative potential, strikingly illustrated in transplantation experiments using limited number of cells. In mice, serial transplantation experiments suggest that individual hematopoietic cells are capable of extensive self‐renewal and that any possible limitations in the replicative potential of individual hematopoietic stem cells are not affecting normal blood cell formation. The situation with human hematopoietic stem cells is less clear. Unlike the situation in the mouse, the telomere length in nucleated human blood cells shows a remarkable decline with age. Furthermore, even partial telomerase deficiency in humans typically results in marrow failure, whereas complete lack of telomerase is tolerated up to several generations in the mouse. The decline in telomere length in human leukocytes with age follows a cubic function and is much higher in lymphocytes than in granulocytes. This finding suggests that, under normal circumstances, telomere loss is more likely to compromise the function of lymphocytes than the function of hematopoietic stem cells. To reconcile differences in telomere biology between man and mice, it has been proposed that telomere shortening evolved as a tumor suppressor mechanism in long‐lived species that may not exist in shorter‐lived mammals. According to this model, telomeres in human cells are intimately involved in signaling cell cycle progression and cell division. Most likely, a minimum number of telomere repeats is required at each telomere to prevent activation of a “telomere checkpoint” and allow cell cycle progression. Telomere length measurements appear useful to distinguish between depletion and exhaustion of hematopoietic stem cells as a cause of marrow failure.
    Materialart: Online-Ressource
    ISSN: 0077-8923 , 1749-6632
    URL: Issue
    RVK:
    Sprache: Englisch
    Verlag: Wiley
    Publikationsdatum: 2003
    ZDB Id: 2834079-6
    ZDB Id: 211003-9
    ZDB Id: 2071584-5
    SSG: 11
    Standort Signatur Einschränkungen Verfügbarkeit
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  • 9
    In: Science, American Association for the Advancement of Science (AAAS), Vol. 288, No. 5466 ( 2000-04-28), p. 665-669
    Kurzfassung: The potential of cloning depends in part on whether the procedure can reverse cellular aging and restore somatic cells to a phenotypically youthful state. Here, we report the birth of six healthy cloned calves derived from populations of senescent donor somatic cells. Nuclear transfer extended the replicative life-span of senescent cells (zero to four population doublings remaining) to greater than 90 population doublings. Early population doubling level complementary DNA-1 (EPC-1, an age-dependent gene) expression in cells from the cloned animals was 3.5- to 5-fold higher than that in cells from age-matched (5 to 10 months old) controls. Southern blot and flow cytometric analyses indicated that the telomeres were also extended beyond those of newborn ( 〈 2 weeks old) and age-matched control animals. The ability to regenerate animals and cells may have important implications for medicine and the study of mammalian aging.
    Materialart: Online-Ressource
    ISSN: 0036-8075 , 1095-9203
    RVK:
    RVK:
    Sprache: Englisch
    Verlag: American Association for the Advancement of Science (AAAS)
    Publikationsdatum: 2000
    ZDB Id: 128410-1
    ZDB Id: 2066996-3
    ZDB Id: 2060783-0
    SSG: 11
    Standort Signatur Einschränkungen Verfügbarkeit
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  • 10
    Online-Ressource
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    Oxford University Press (OUP) ; 2003
    In:  Journal of Leukocyte Biology Vol. 73, No. 2 ( 2003-02-01), p. 289-296
    In: Journal of Leukocyte Biology, Oxford University Press (OUP), Vol. 73, No. 2 ( 2003-02-01), p. 289-296
    Kurzfassung: To address questions about telomere length regulation in nonhuman primates, we studied the telomere length in subpopulations of leukocytes from the peripheral blood of baboons aged 0.2–26.5 years. Telomere length in granulocytes, B cells, and subpopulations of T cells all decreased with age. Overall, telomere length kinetics were lineage- and cell subset-specific. T cells showed the most pronounced, overall decline in elomere length. Levels of telomerase in stimulated T cells from old animals were lower than in corresponding cells from young animals. Memory T cells with very short telomeres accumulated in old animals. In contrast, the average telomere length values in B cells remained relatively constant from middle age onward. Individual B cells showed highly variable telomere length, and B cells with very long telomeres were observed after the ages of 1–2 years. In general, cell type-specific telomere kinetics in baboons were remarkably similar to those observed in humans.
    Materialart: Online-Ressource
    ISSN: 0741-5400 , 1938-3673
    RVK:
    Sprache: Englisch
    Verlag: Oxford University Press (OUP)
    Publikationsdatum: 2003
    ZDB Id: 2026833-6
    SSG: 12
    Standort Signatur Einschränkungen Verfügbarkeit
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