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  • 1
    ISSN: 1476-4687
    Source: Nature Archives 1869 - 2009
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
    Notes: [Auszug] In non-autoradiographic procedures, labels are either coupled directly to the probe6'7 or introduced via specific antibodies after hybridization with haptenized probes2'8. The procedure described here is an example of the latter, in which the nucleic acids are chemically modified with ...
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  • 2
    ISSN: 1432-119X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary A non-radioactive in situ hybridization technique is described which allows the simultaneous detection of different DNA sequences. To demonstrate the feasibility of the proccdure, metaphases and interphase nuclei of a human-mouse somatic cell hybrid were simultaneously hybridized with mercurated total human DNA and a biotinylated mouse satellite DNA probe. After the hybridization, the probes were detected immunocytochemically using two different and independent affinity systems. By this approach we visualized the two DNA target sequences in metaphase chromosomes and in interphase nuclei with FITC and TRITC fluorescence, or blue (alkaline phosphatase) and brown (peroxidase) precipitated enzyme products. This method not only allows detection of intact chromosomes but also the visualization of rearrangements between parts of human and mouse chromosomes. Furthermore, the technique demonstrates the high topological resolution of nonradioactive in situ hybridizations.
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  • 3
    Electronic Resource
    Electronic Resource
    Springer
    Histochemistry and cell biology 86 (1987), S. 609-615 
    ISSN: 1432-119X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary A number of immunocytochemical detection systems for determining the chromosomal localization of specific nucleic acid sequences by non-radioactive in situ hybridization have been compared. The procedures were: 1. the peroxidase/diaminobenzidine (PO/DAB) combination, either or not gold/silver intensificated; 2. alkaline phosphatase marking using the nitro-blue tetrazolium plus bromochloro-indolyl phosphate substrate combination (AP/NBT+CIP); and 3. immunogold with or without silver enhancement. The procedures were first tested and optimized in dot blot experiments and then applied to in situ hybridization. As hybridization probes, both a middle-repetitive and a unique sequence (modified with 2-acetyl-aminofluorene (AAF)) were used. The advantages and dis-advantages of the various methods for reflection contrast (RC) or transmission electron microscopic (TEM) visualization of hybrids are discussed.
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  • 4
    ISSN: 1432-0584
    Keywords: Key words Differential display ; Myelodysplastic syndromes ; Chromosomal aberration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  The most common chromosomal aberrations in myelodysplastic syndromes (MDS) are complete or partial loss of chromosomes 5 and 7, and trisomy 8. To identify genes important in the pathogenesis of this disease that could be associated with these gross chromosomal defects, we have employed the differential display PCR (DDPCR) procedure developed by Liang and Pardee. This method allows simultaneous comparison of several cDNA sources for the presence of differentially expressed genes. Polymorphonuclear cells (PMNs) from two MDS patients, containing a 5q deletion or a trisomy 8, and three healthy controls were used. Initial screening resulted in the identification of five and three partial cDNA sequences, respectively that were either differentially expressed in both patient samples or in individual patients, as compared with the controls. The authenticity of aberrant expression was verified by reanalyzing the same primer combinations on newly prepared cDNA. Differential expression of the three remaining fragments was subsequently checked on a larger panel of MDS patients, using amplicon-specific primer sets. These were obtained by cloning and sequencing of the fragments. For one partial cDNA (DC3), the original expression pattern, i.e., decreased expression in individual MDS patients, was confirmed. These results demonstrate the utility of the DDPCR procedure to isolate differentially expressed sequences in primary patient samples where the availability of cells is a limiting factor.
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  • 5
    ISSN: 1432-0584
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Conclusions During the past 10 years, MDS has become a topic of considerable interest due to the development of new cytogenetic and molecular techniques and the introduction of the clinical administration of hematopoietic growth factors. The cytological and clinical classifications of the diseases comprising the syndrome are based on two main subtypes, those with a low risk of acquiring acute leukemia and those with a high risk. Which one will prove to be the simplest and most reliable classification system for the individual patient is not yet clear. Studies on clonality have shown that the myeloid cells in most of the patients studied thus far are monoclonal in nature. These methods can be used to evaluate the effect of treatment regimens. The kinetics of the in vitro growth of hematopoietic precursor cells from MDS patients is abnormal, with suboptimal colony formation. Cultures grown in the presence of certain growth factors show preferential growth of normal hematopoietic cells, which may be of clinical relevance in the future. Administration to MDS patients of hematopoietic growth factors such as GM-CSF, G-CSF, and IL-3 improves neutrophil counts in the majority of cases. The effect of erythropoietin is disappointing in most cases. At present, polyclonal hematopoiesis is rarely induced by growth factor treatment alone. More recently identified growth factors, such as MGF, might preferentially affect normal hematopoiesis at the expense of the preleukemic clones, thus changing the natural course of the disease.
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  • 6
    ISSN: 1432-0584
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Type of Medium: Electronic Resource
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  • 7
    ISSN: 1432-0584
    Keywords: AraC and DAC resistance ; dck gene ; Inactivating mutations
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The deoxycytidine kinase (dck) gene encodes the enzyme responsible for the metabolic activation of the antileukemic drugs cytosine arabinoside (AraC) and 5-aza-2′-deoxycytidine (decitabine, DAC). Thedck locus was analyzed at the chromosomal and the molecular level in a model of rat leukemic cell lines, in which AraC and DAC resistance was induced, that was marked bydck deficiency. At the chromosomal level, karyotype analysis of metaphase spreads revealed the presence of an aberrant 2q+ chromosome in the AraC-resistant cell line and a (Xq;11q) translocation in its subclone RA/7. The DAC-resistant lines were identical to the parental RCL/O. Fluorescence in situ hybridization on normal rat fibroblast metaphase spreads localized the ratdck gene to chromosome 14q21-q22, a region that was not involved in any of the observed karyotypic aberrations. Analysis at the molecular level revealed an identical rearrangement of thedck gene in the AraC-resistant cell line RCL/A and its subclone RA/7 that resulted in the absence ofdck expression, as assessed by RT-PCR. No genomic rearrangements were observed in a DAC-resistant cell line RCL/D or in its subclone RD/1. However, detection of a single-stranded conformation polymorphism (SSCP) allowed the identification of a single C to G substitution (His to Gln) in thedck cDNA of the DAC-resistant RD/1 clone. The data demonstrate that exposure to AraC and DAC induces a resistant phenotype marked by functionaldck deficiency that may be the consequence of mutations occurring in thedck gene.
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  • 8
    ISSN: 1432-0584
    Keywords: Key words Hematopoiesis ; Hematopoietic growth factors ; Cytokines ; Appendicitis ; Bone marrow
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  To analyze the role of hematopoietic growth factors (HGFs) and other cytokines in the regulation of hematopoiesis in vivo, we investigated HGFs and cytokine gene expression in appendices obtained from patients who underwent surgery for suspected appendicitis. Concomitantly, HGF gene expression was studied in bone marrow (BM) biopsy specimens and plasma HGF levels were measured. G-CSF gene expression was detected in inflamed but not in normal appendices. With one exception, GM-CSF was detectable in all appendices whether inflamed or not, whereas IL-3, except for one case, was not expressed in appendices. None of the investigated HGFs appeared to be expressed in BM biopsy specimens concurrently obtained with the appendices. Plasma G-CSF levels were significantly elevated in patients with appendicitis compared with patients without inflamed appendices. Circulating levels of GM-CSF and IL-3 were not increased. Significant up-regulation of IL-8 and IL-6 gene expression was observed in response to inflammation, in contrast to IL-1α and IL-1β expression, which appeared not to be influenced by the inflammatory state. These data indicate that G-CSF, and not GM-CSF or IL-3, is essential for the regulation of inducible granulopoiesis in acute inflammatory conditions, and that G-CSF acts in an endocrine fashion.
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  • 9
    ISSN: 1432-0584
    Keywords: Cytokines ; Monocytes ; GM-CSF ; IL-2 ; IL-3 ; IL-4
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary IL-4, a product of the T-helper 0 (Th0) and 2 (Th2) subset, was originally described as a B-cell stimulatory factor and has subsequently been found to suppress IL-1α, IL-1Β, IL-6, IL-8, and TNF-α gene expression in monocytes stimulated with LPS, and to upregulate IL-1 receptor antagonist (IL1-RA) gene expression. In this study we investigated the effect of IL-4 on the expression of cytokine genes in monocytes evoked by other T-helper cell cytokines: IL-2, IL-3, and GM-CSF. IL-4 down-regulated mRNA accumulation of the proinflammatory cytokines IL-1Β, IL-8, and TNF-α in monocytes stimulated with IL-2, IL-3, and GM-CSF. IL-4 also suppressed the IL-2-induced IL-6 mRNA expression. Temporal analysis of the IL-4 down-regulatory effect on the IL-2-, IL-3-, or GM-CSF — induced proinflammatory cytokine gene expression in monocytes provided evidence that IL-4 acts predominantly oh the post-transcriptional level. This was supported by the observation that the down-regulatory capacity of IL-4 appeared to be dependent on de novo protein synthesis. IL-4 did not exert significant influence on the induction of expression of IL-1-RA or various CSFs by IL-2, IL-3, and GM-CSF. We hypothesize that owing to its being a down-regulator of the expression of proinflammatory cytokines induced by IL-2, IL-3, and GM-CSF — products of activated ThO or Th1 cells —together with its capacity as a B-cell stimulatory factor, IL-4 has an important role in directing the immune response.
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  • 10
    ISSN: 1432-0584
    Keywords: Key words AraC and DAC resistance ; dck gene ; Inactivating mutations
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  The deoxycytidine kinase (dck) gene encodes the enzyme responsible for the metabolic activation of the antileukemic drugs cytosine arabinoside (AraC) and 5-aza-2′-deoxycytidine (decitabine, DAC). The dck locus was analyzed at the chromosomal and the molecular level in a model of rat leukemic cell lines, in which AraC and DAC resistance was induced, that was marked by dck deficiency. At the chromosomal level, karyotype analysis of metaphase spreads revealed the presence of an aberrant 2q+ chromosome in the AraC-resistant cell line and a (Xq;11q) translocation in its subclone RA/7. The DAC-resistant lines were identical to the parental RCL/O. Fluorescence in situ hybridization on normal rat fibroblast metaphase spreads localized the rat dck gene to chromosome 14q21–q22, a region that was not involved in any of the observed karyotypic aberrations. Analysis at the molecular level revealed an identical rearrangement of the dck gene in the AraC-resistant cell line RCL/A and its subclone RA/7 that resulted in the absence of dck expression, as assessed by RT-PCR. No genomic rearrangements were observed in a DAC-resistant cell line RCL/D or in its subclone RD/1. However, detection of a single-stranded conformation polymorphism (SSCP) allowed the identification of a single C to G substitution (His to Gln) in the dck cDNA of the DAC-resistant RD/1 clone. The data demonstrate that exposure to AraC and DAC induces a resistant phenotype marked by functional dck deficiency that may be the consequence of mutations occurring in the dck gene.
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