Routine fluorescence in situ hybridization analysis for detection of BCR-ABL rearrangement in myeloproliferative disorders.
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Chromosomes, Human, Pair 22↗
Biomedical subjects
L Stuppia
Publications and source records attributed to L Stuppia.
Clustering of Y chromosome deletions in subinterval E of interval 6 supports the existence of an oligozoospermia critical region outside the DAZ gene.
Y chromosome molecular analysis was performed using the STS-PCR technique in 50 patients with oligozoospermia. Microdeletions of interval 6 of the Y chromosome were detected in seven patients, in six of whom subinterval E was affected. All patients retained the RBM1 and DAZ genes, while in one deletion involved the SPGY gene. The size of the deletion was not apparently related to the severity of the disease. These results suggest the presence of an oligozoospermia critical region on the Y chromosome within subinterval E of interval 6.
Adult↗
Complex chromosome translocations of standard t(8;21) and t(15;17) arise from a two-step mechanism as evidenced by fluorescence in situ hybridization analysis.
The authors report the results of cytogenetic and fluorescence in situ hybridization (FISH) analysis performed on complex chromosome translocations (CCTs) of t(8;21) and t(15;17) standard translocations associated with two M2 subtypes of acute myeloid leukemia (AML-M2) and four acute promyelocytic leukemia (APL), respectively. In one of two AML-M2 patients FISH analysis showed part of chromosome 21 on the der(8) and material from this chromosome on the der(21) and on chromosome 1 at band p32, suggesting that the t(8;21) occurred as the primary step. In the second AML-M2 patient. FISH displayed part of chromosome 21 on the der(8) and material from this chromosome on the der(21) but not on the third rearranged chromosome. Therefore, it is unclear whether chromosome 2 was rearranged secondary to the standard t(8;21). In four APL patients, FISH analysis showed material derived from chromosome 17 on the der(15). Moreover, in two patients with an i(17q) FISH disclosed material from chromosome 15 at the ends of both arms of the i(17q), suggesting that it occurred after the standard t(15;17). In the remaining two APL patients, FISH showed material from chromosome 15 on the der(17) and on chromosome 21 at band q22 in one case, and material of the p arm of chromosome 17 on chromosome 4 at band q11 in the other, demonstrating that in these two cases the first mutation also had been the t(15;17). Therefore, FISH analysis revealed that CCTs in five patients were secondary changes which occurred after standard t(8;21) and t(15;17), thus clarifying the hierarchy of the cytogenetic events, their role in the pathogenesis of the disease, and the associated clinic-hematologic findings.
Adult↗
Human immunodeficiency virus type 1 tat protein modulates fibronectin expression in thymic epithelial cells and impairs in vitro thymocyte development.
Infection of both lymphoid and stromal components of the thymus by human immunodeficiency virus type 1 (HIV-1) suggests that impairment of lymphocyte differentiation from early T cells progenitors in the thymus may contribute to the HIV-induced T cell depletion. Cross-talk between immature thymocyte and thymic epithelium through cell-to-cell adhesion mediated by fibronectin/receptor interaction plays a central role in driving T cell development. HIV-1 tat protein, like fibronectin, contains an RGD sequence involved in the interaction with fibronectin receptor. We demonstrated that gene transfer-mediated tat expression in thymic stroma is able to influence the in vitro maturation of T cell progenitors as tat-expressing epithelial cells have a decreased ability to drive the generation of CD4+8+ thymocytes from CD4-8- precursors. Furthermore, tat-expressing cells produce more fibronectin and display upregulation of VLA-5 cell surface receptor levels compared to control cells, while alpha v expression was unchanged. Cellular distribution of fibronectin is also influenced by tat. Fibronectin is distributed in the whole cell surface and along cell processes in control cells whereas it is mainly concentrated in the intracytoplasmic area in tat-expressing cells. Therefore, expression of tat in thymic epithelial cells impairs thymocyte maturation and modulates fibronectin expression: this suggests a crucial role of this viral protein in regulating the T lymphocyte differentiation program through modulation of intrathymic lympho-stromal interactions.
Animals↗
Molecular studies in three patients with isodicentric Y chromosome.
Three patients carrying an isodicentric (idic) Y chromosome associated with a mosaic 45,X cell line were studied using molecular techniques. Genotype-phenotype correlations suggested an effect of the 45,X cell line on sexual differentiation. A relationship was established between instability of the idic(Y) chromosome and localization of the breakpoint on Yq, and between azoospermia and deletion of interval 6 on Yq.
Adult↗
Microdeletions in interval 6 of the Y chromosome detected by STS-PCR in 6 of 33 patients with idiopathic oligo- or azoospermia.
It has been proposed that interval 6 of the human Y chromosome contains the gene or genes that control spermatogenesis (AZF, azoospermia factor). We have studied this region in 33 patients with oligo- or azoospermia, using PCR amplification of the YRRM1 (RBM1) gene and of 13 sequence-tagged sites (STSs), all mapping within interval 6. Six of the 33 patients showed no amplification of specific STSs, whereas there was no failure of amplification in normal male controls. We deduce that these six patients had microdeletions in interval 6 of the Y chromosome that correlated with the oligo- or azoospermia of these individuals. On biopsy of the testis, two of these patients showed a low number of germ cells, and four showed arrest with spermatides. We conclude that PCR amplification of Y-specific regions is a powerful and very sensitive tool for screening infertile men.
Adult↗
Morphometric and functional study of apoptotic cell chromatin.
Apoptosis is usually characterized by profound morphological nuclear changes. Chromatin undergoes a progressive condensation that eventually involves all the nucleus. At earlier stages chromatin appears as divided in compact and diffuse areas, while the nuclear pores disappear from the nuclear envelope that surrounds the compact areas, and cluster around diffuse chromatin. Here we have performed a morphometric study on the different chromatin areas of freeze-fractured apoptotic cell nuclei in order to investigate its morphometric and functional organization. We have found large portions of inactive chromatin aggregations corresponding to the dense cap-shaped patches, while domains of nucleosomic fibres have been identified in the diffuse chromatin areas. The correlation of the nucleosomic fibre/diffuse chromatin domain with the nuclear pore clusters is demonstrated, and its implications with a possible residual nuclear activity are discussed.
Journal Article↗
FISH detection of mixed chimerism in 33 patients submitted to bone marrow transplantation.
Fluorescence in situ hybridization (FISH) and cytogenetic analysis were carried out in 33 transplanted patients suffering from different hematologic disease using probes for X and Y chromosomes and ABL and BCR genes. FISH showed that recipient cells were invariably present during post-transplant follow-up. Stable minimal residual disease was associated with clinical and hematologic remission, while a progressive increase of host cells was strictly related with disease relapse. Cytogenetic investigation on the same samples showed recipient cells only in few cases. It was concluded that FISH analysis is useful for: (1) characterizing cases in which standard cytogenetic analysis has failed; (2) detecting host cells in sex-mismatched transplanted patients; and (3) evaluating Ph-negative CML with the BCR/ABL rearrangement. The possibility of detecting chromosome rearrangements in interphase nuclei using FISH analysis improves diagnosis and prediction of disease evolution and prompts earlier therapeutic approaches.
Adolescent↗
Multiple fluorescence and reflectance simultaneous detection by confocal microscopy of HaeIII digested DNA sequences.
Human metaphase chromosomes were isolated and digested in situ with HaeIII restriction enzyme to detect cytosine and guanine-rich sequences (CpG islands), which are known to be associated with most of the mammalian genes. Digested DNA was reconstructed by in situ nick translation employing digoxigenin-labeled nucleotides. The DNA sequences were revealed by antibodies conjugated either with fluorescein isothiocyanate or 1-nm colloidal gold. DNA was counterstained with propidium iodide. A sensitive, high resolution method for visualizing three signals, simultaneously excited by a single argon laser line of 488 nm has been developed. The green fluorescence of fluorescein isothiocyanate was detected in combination with the red fluorescence of propidium iodide, and the third signal was imaged by employing the reflectance mode of the confocal microscope after silver enhancement of the gold beads. The high reflectance intensity, the accurate localization and the non-fading properties of colloidal gold made the reaction a valuable tool for the detection of antigens and, as a consequence, of specific DNA sequences in chromosome preparations. Overlaying of three signals allowed the simultaneous observation of distinct structures: total DNA, as well as fluorescein- and gold-labeled sequences after in situ nick translation, or total DNA and centromeric sequences of two different chromosome pairs (17 and X) after in situ hybridization. The use of HaeIII restriction enzyme that cut CpG islands combined with in situ nick translation identified the chromosome sites where active, inactive or housekeeping genes can be located. In chromosomes, the fluorescent reaction pattern showed large areas of labeling, while a more defined staining, often organized in spot pairs that resembled an R-like banding, was detected when the reflected mode was used. These results are confirmed by the observation that R-like bands actually are multiple symmetrical spots localized on sister chromatids. In addition, some chromosomes, and in particular 1 and 9, displayed a C-negative banding due to the negativity of the centromeric areas. Reflectance confocal scanning microscopy and in situ nick translation represent a powerful tool to study the in situ genome organization.
Chromosomes, Human↗
Widening of a Y-chromosome interval-6 deletion transmitted from a father to his infertile son accounts for an oligozoospermia critical region distal to the RBM1 and DAZ genes.
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Adult↗
Different approaches to the study of apoptosis.
The morphological features of cell undergoing programmed cell death is well known and has been widely described in a number of experimental models with a variety of apoptotic triggering agents. Despite the similar cell behaviour, underlying molecular events seem variable and only partially understood. A multiple approach appears crucial to better clarify the phenomenon. The first technique, DNA gel electrophoresis, allows the identification of fragmented DNA and has been long considered the hallmark of apoptosis. Different patterns of DNA cleavage, which can be identified by conventional or "pulsed-field gel" electrophoresis, are presented and discussed. "In situ" labelling methods are also described both with terminal deoxynucleotidyl transferase and DNA polymerase I, aimed at the study of the distribution of DNA cleavage areas. Flow cytometry is also proposed and different technical approaches, based on different laser utilizations, are discussed. Ultrastructural analysis, allowing the study of apoptotic cell details, is finally considered.
Animals↗
Nuclear translocation of beta II PKC isoenzyme in phorbol ester-stimulated KM-3 pre-B human leukemic cells.
Members of the protein kinase C (PKC) family play a key role in regulating cell growth and differentiation in response to several stimuli, including hormones, neurotransmitters, and growth factors. The different properties and substrate specificity of the PKC isoforms are not fully understood, and they are assumed to have specific functions in intracellular signaling. In lymphoid cells, the effects of PMA and Ca2+ ionophore, singly or in combination, on activation and expression of Ca(2+)-dependent PKC at the level of protein and messenger RNA have been examined. Starting from these observations and the possibility that differential isoenzyme expression might contribute to the differences in phorbol ester sensitivity of lymphoid cells, it seemed worthwhile to investigate the expression and the modulation of PKC isoforms in KM-3 cells, a human pre-B cell line, upon treatment with phorbol 12-myristate 13-acetate (PMA). Using multiparametric analysis we detected three PKC isoforms in the KM-3 cell line: alpha, beta II, and zeta. PMA treatment causes an intranuclear translocation of the beta II isoform, via the nuclear pore complex, associated with the interchromatinic regions. These data suggest that the beta II isoenzyme may play a strategic role in signal transduction and regulation of specific gene expression in B lymphocytes.
Biological Transport↗
Chromosome mapping of the rat phospholipase C beta 1 gene.
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Animals↗
Retrospective investigation of hematopoietic chimerism after BMT by PCR amplification of hypervariable DNA regions.
We report on nine patients submitted to BMT with sex-matched donors and investigated by means of PCR amplification of the VNTRs ApoB, D1S80, DXS52, and D17S5. In all cases it was possible to detect a polymorphism able to distinguish between donor and patient cells, thus allowing us to recognize the presence of complete or mixed chimerism. In eight patients PCR analysis showed a complete chimerism during the entire follow-up. Only one of these patients relapsed, while the others are alive and without any sign of relapse 56.2 months (mean) after BMT. Mixed chimerism was detected in only one patient, who relapsed 3 months after this finding. These results confirm the usefulness of the study of PCR-amplified VNTRs in the assessment of marrow engraftment after BMT, mostly in sex-matched transplants where, in the absence of specific chromosome rearrangements, cytogenetic or FISH analysis cannot be used.
Adolescent↗
Epidermal growth factor promotes a neural phenotype in thymic epithelial cells and enhances neuropoietic cytokine expression.
Neural crest-derived cells populate the thymus, and their coexistence with epithelial cells is required for proper organ development and T cell education function. We show here that epidermal growth factor (EGF), a major epithelial cell growth-enhancing agent, has a morphogenetic action to promote the expression of a neuronal phenotype (e.g., neurofilament expression) in cultured thymic epithelial cells that are characterized by a cytokeratin-positive epithelial cell background. The proliferation of such neurodifferentiated cells is also enhanced by EGF. Furthermore, the growth factor enhances cells that express the genes encoding the preprotachykinin A-generated neuropeptides and bipotential neuropoietic and lymphopoietic cytokines ciliary neurotrophic factor and interleukin-6. These cytokines also enhance the neuronal phenotype of thymic epithelial cells. Therefore, EGF appears to be a composite autocrine/paracrine neuromodulator in thymic stroma. This suggests that EGF may regulate thymus-dependent immune functions by promoting neuronal gene expression in neural crest-derived cells.
Animals↗
Duplication Xp22.2 and pseudoisodicentric Yq detected by FISH and PCR in a sterile male.
A chromosome mosaicism with two cell lines was diagnosed in a sterile man. One cell line had a 45, -Y, dup (X) (p22.2) karyotype and accounted for 83% of lymphocytes analyzed. Fluorescence in situ hybridization (FISH) analysis with specific X and Y probes excluded a translocation between the short arms of the X and Y chromosomes and showed that Xp duplication involved a region containing the DXS85 locus, distal to the ZFX and DSS sites. The other cell line consisted of a diploid karyotype with a rearranged Y chromosome, which was shown to be a pseudoisodicentric Yq by FISH. Moreover, FISH with a specific probe for the AZF locus and polymerase chain reaction using Yq SY108 and SY121 primers showed no signals for this region, possibly accounting for the azoospermia in this patient.
Adult↗
Molecular characterization of an unusual variant t(15;17) detected in an APL patient.
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Adult↗
Chromosome mapping of the human arrestin (SAG), beta-arrestin 2 (ARRB2), and beta-adrenergic receptor kinase 2 (ADRBK2) genes.
Two types of proteins play a major role in determining homologous desensitization of G-coupled receptors: beta-adrenergic receptor kinase (beta ARK), which phosphorylates the agonist-occupied receptor and its functional cofactor, beta-arrestin. Both beta ARK and beta-arrestin are members of multigene families. The family of G-protein-coupled receptor kinases includes rhodopsin kinase, beta ARK1, beta ARK2, IT11-A (GRK4), GRK5, and GRK6. The arrestin/beta-arrestin gene family includes arrestin (also known as S-antigen), beta-arrestin 1, and beta-arrestin 2. Here we report the chromosome mapping of the human genes for arrestin (SAG), beta-arrestin 2 (ARRB2), and beta ARK2 (ADRBK2) by fluorescence in situ hybridization (FISH). FISH results confirmed the assignment of the gene coding for arrestin (SAG) to chromosome 2 and allowed us to refine its localization to band q37. The gene coding for beta-arrestin 2 (ARRB2) was mapped to chromosome 17p13 and that coding for beta ARK2 (ADRBK2) to chromosome 22q11.
Antigens↗