Malformaciones renoureterales en distintas variantes del síndrome de Turner / Renoureteral malformations in different variants of Turner`s syndrome

Se estudió, através de la realización de un urograma descendente, la incidencia de malformaciones renoureterales en 33 pacientes con el síndrome de Turner o algunas de sus variantes. Se comprobó la existencia de algún tipo de anomalía en 23 casos (69,7 %) y sólo en 10 el estudio fue normal. Las alteraciones más frecuentes fueron: pelvis renal doble (30,3 %). malrotación renal (15,2 %) y riñón en herradura (12,1 %). No se halló una asociación directa entre la fórmula cromosómica y las malformaciones renoureterales encontradas, pero si se observó que las malformaciones más graves ocurren con mayor frecuencia en las pacientes con monosomía X. Se recomienda el estudio sistemático del aparato renoureteral en toda paciente con el diagnóstico de síndrome de Turner o algunas de sus variantes

Digoxigenin-labeled probes can detect single-copy genes in human metaphase chromosomes.

A technique of in situ hybridization on metaphases of chromosomes by a digoxigenin-labeled probe is described. This technique was able to detect single DNA sequences of 2 and 7 kilobases. The results obtained were compared with those of a biotin streptavidin alkaline phosphatase-based detection system. The digoxigenin method was at least as efficient and sensitive as the biotin-streptavidin method.

Intermediate structures in nuclear morphogenesis following metaphase from HeLaS3 cells can be isolated and temporally grouped.

Previously nuclear reformation following metaphase in HeLaS3 cells was conceptualized in terms of a stepwise process which was continuous throughout anaphase and telophase. This concept was based on a three-dimensional visualization by scanning electron microscopy (SEM) of individual, organically prepared chromatid structures (prenuclei) which could be sequentially arranged. Morphologic analysis revealed unique topographies and morphometric properties which suggested that it should be possible to isolate populations of prenuclei aqueously. Such an isolation using detergents and density centrifugation is presented which yields metaphase plates and two populations of prenuclei with distinctive morphology. Essentially, prenuclei are freed from late mitotic cells in suspension cultures of synchronized HeLaS3 cells by treatment with 0.1% Nonidet-P40 followed by treatment with a mixture of Tween 40-desoxycholate (0.5%). Critical for the isolation is the presence of a divalent cation (5 mM Mg(+)+) and an acid pH (approximately 5.8). After density centrifugation, 2N decondensing structures (late intermediates) are recovered from 42% Percoll, and a mixture of 2N predecondensing (early intermediates) and 4N metaphase plates are recovered from 52% Percoll. The latter intermediates can be further separated into highly enriched populations (greater than 94% pure) by fluorescence-activated sorting. Predecondensing structures are of the same overall morphology as prenuclei isolated previously by organic means, can also be ordered sequentially to demonstrate nuclear morphogenesis, and retain centromere/kinetochore loci. These chromosomal loci based on immunostaining of individual structures appear to be positioned centrally during chromatid reassociation and then appear to be dispersed prior to structural rearrangements leading to formation of a disc-like prenucleus.(ABSTRACT TRUNCATED AT 250 WORDS)

Unfixed and fixed human chromosomes show different staining patterns after restriction endonuclease digestion.

Restriction endonucleases (REs) have been widely used to produce banding patterns on chromosomes, but it remains uncertain to what extent the patterns are due to the sequence specificity of the enzymes, and to what extent chromatin structure influences the pattern of digestion. To throw light on this question, we have digested with restriction endonucleases unfixed chromosomes prepared in two different ways (isolated, and whole metaphase cells spread with a cytocentrifuge) and compared the results with those obtained on conventionally fixed chromosomes. Unfixed isolated chromosomes are easily destroyed by REs; after fixation with cold methanol, which produced minimal alteration to the chromatin structure, the chromosomes are resistant to the action of REs, and conventional methanol-acetic acid fixation is required to permit the induction of banding patterns by REs. Unfixed cytocentrifuge preparations, in which the chromosomes are still surrounded by cytoplasm, are much more resistant to the action of REs, and again banding patterns were only induced after methanol-acetic acid fixation. We conclude that the action of restriction endonucleases on chromosomes is strongly influenced by chromatin organisation, and that methanol-acetic acid fixation is required to permit the induction of conventional banding patterns on chromosomes.

HPV16 E6 and E7 proteins cooperate to immortalize human foreskin keratinocytes.

The human papillomavirus types (HPVs) most often associated with cancer of the cervix, such as HPV16, have been reported previously to immortalize normal human foreskin keratinocytes in vitro, while the types that are primarily associated with benign cervical lesions failed to do so. In this study we have determined the HPV16 genes that are responsible for the immortalizing activity of the viral genome. Transfection with a plasmid in which E6 and E7 were the only intact open reading frames (ORFs) induced an indefinite life-span in the keratinocytes with an efficiency similar to that of the entire early region of the viral DNA. Mutants in the E6E7 clone with inactivating lesions in E6 or E7 failed to induce immortalization. When transfected alone, E7 could induce hyperproliferation, but these cells eventually senesced. By itself, E6 exhibited no activity, Co-transfection of a plasmid with an intact E6 ORF and a second plasmid with an intact E7 ORF generated keratinocyte lines with indefinite growth potential. The E6 and E7 proteins were detected in the lines induced by the E6E7 DNA and by co-transfection of the E6 and E7 plasmids. Therefore, we conclude that HPV16 E6 and E7 cooperative to immortalize human keratinocytes in vitro. Changes in cellular gene expression are probably also required for immortalization since all of the keratinocyte lines examined were aneuploid. Serum and calcium resistant sublines were isolated from the E6E7 induced lines, indicating that other HPV genes do not play an obligatory role in the generation of resistance to differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)

[A photographic microfluorimetric method for determining the DNA content in individual human chromosomes]. / Fotograficheskii mikrofluorimetricheskii metod opredeleniia soderzhaniia DNK v individual'nykh khromosomakh cheloveka.

A method for determination of DNA contents in individual human chromosomes has been elaborated based of the two step analysis: 1) identification of chromosomes by Q-banding; 2) photographic microfluorimetry of chromosomes after the Feulgen staining (a fluorescent variant using a Schiff-type reagent of Auramine-SO2). The DNA content in 24 human chromosomes was calculated in absolute values (fg). The data obtained are compared with the evidence published elsewhere and provided by different cytophotometric methods.

[Sézary syndrome: morpho-immuno-phenotypic, cytogenetic and molecular characterization]. / Síndrome de Sézary: caracterización morfoinmunofenotípica, citogenética y molecular.

The immunologic, cytogenetic and molecular data from a patient with Sézary syndrome are reported. Immunohistochemical analysis disclosed 90% helper CD4 cells and 10% CD8 cells. One of the most significant cytogenetic abnormalities was a t(7; 14) translocation at the level of the 7p13-15 and 14q11 bands, just where the T-cell receptors are located. At the molecular level, rearrangements of the alpha (in 14q11 chromosome), beta (in the 7q32) and gamma (in the 7p15) receptors were found. The translocation between chromosomes 7 and 14, at the level of the mentioned bands, could be responsible for some of the rearrangements found at the molecular level.

Hallazgos cromosómicos en parejas susceptibles al diagnóstico prenatal citogenético / Chromosomal findings in couples susceptible to cytogenetic perinatal diagnosis

En la Ciudad de la Habana, paralelamente con el programa de Diagnóstico Prenatal Citogenético, se realizan estudios cromosómicos en sangre periférica a las parejas que presentan hijos con síndrome de Down, abortos espontáneos recurrentes, hijos previos con malformaciones múltiples y otros. Se presentan los resultados de los análisis cromosómicos realizados en el año 1986 y la relación de estos pacientes con el Diagnóstico Prenatal Citogenético. Se estudiaron 164 pacientes en los cuales se encontró 1 aberración numérica, 1 estructural y 5 variantes cromosómicas normales

Identification of a deoxyribonucleic acid allelic variant for beta 1-4 galactosyltransferase expression associated with male sperm binding/penetration infertility.

Studies on mouse sperm-egg binding and fertilization have been suggested to involve the interaction of sperm-associated beta 1-4 galactosyltransferase with egg zona pellucida glycoproteins. A population of human males, whose sperm demonstrated an inability to penetrate ovulated zona pellucida-free hamster eggs in vitro, were examined for the level of activity of beta 1-4 galactosyltransferase. The level of enzyme activity was found to be reduced in human sperm isolated from this group of individuals compared with a known hamster penetration-positive group. Analysis of the deoxyribonucleic acid from these individuals by Southern hybridization with a putative human complementary deoxyribonucleic acid clone to beta 1-4 galactosyltransferase identified a unique allele lacking 0.8 and 0.4 kb restriction fragments on digestion with the endonuclease Taq I. These results represent the first evidence to suggest that mutations could be associated with the human gene for galactosyltransferase. Our data help to clarify one of the possible molecular mechanisms responsible for sperm-egg binding/penetration interactions.

Alphoid satellite DNA is tightly associated with centromere antigens in human chromosomes throughout the cell cycle.

In this study, we have examined a DNA element specific to the centromere domain of human chromosomes. Purified HeLa chromosomes were digested with the restriction enzyme Sau3AI and fractionated by sedimentation through a sucrose gradient. Fractions showing antigenecity to anticentromere (kinetochore) serum obtained from a scleroderma CREST patient were used to construct a DNA library. From this library we found one clone which has specifically hybridized to the centromere domain of metaphase chromosomes using a biotinylated probe DNA and FITC-conjugated avidin. The clone contained a stretch of alphoid DNA dimer. To determine precisely the relative location of the alphoid DNA stretch and the centromere antigen, a method was developed to carry out in situ hybridization of DNA and indirect immunofluorescent staining of antigen on the same cell preparation. Using this method, we have found perfect overlapping of the alphoid DNA sites with the centromere antigen sites in both metaphase chromosomes and nuclei at various stages in the cell cycle. We have also observed this exact correlation at the attachment sites of artificially extended sister chromatids. These results suggest the possibility that alphoid DNA repeats are a key component of kinetochore structure.

Chromosome banding analysis by slit-scan flow cytometry.

We have investigated the use of fluorescence banding patterns for the resolution of metaphase chromosomes by slit-scan flow cytometry. Fluorescence scans of R-banded chromosomes have been obtained for the entire human karyotype. Metaphase chromosomes were R-banded in suspension by staining with chromomycin A3 after hypotonic treatment in Ohnuki's buffer. Specific fluorescent landmark bands were detected for human chromosomes 1-12. Scans obtained for chromosomes 13-22 did not contain sufficient information for classification. Characteristic fluorescence patterns for human chromosomes 1 and 3 provided the clearest evidence for the detection of R-bands by slit-scan flow cytometry. Specific patterns were detected for human chromosomes 9-12 in which the number and placement of the fluorescent bands served as classifiers.

Analysis of bivariate flow karyotypes.

Bivariate flow karyotype analysis is performed using data from chromosomes stained with two fluorescent dyes, typically chromomycin A3 and Hoechst-33258, and measured in a flow cytometer or cell sorter (Carrano et al.: Proceedings of the National Academy of Sciences of the United States of America 76:1382-1384, 1979; Gray et al.: Proceedings of the National Academy of Sciences of the United States of America 72:1231-1234, 1975; Langlois et al.: Proceedings of the National Academy of Sciences of the United States of America 79:7876-7880, 1982). In the resulting bivariate histogram, most chromosome types appear as individual peaks. In sorting of chromosomes to purify a specific chromosomal type, its corresponding peak in the bivariate histogram is delineated by a rectangular region which surrounds it. All events (objects) that fall within this region trigger the sorting process. In most cases, peaks for different chromosomal types overlap to some extent, and in addition there is always an underlying background due to chromosome fragments and clumps. Thus the sorted population will not be pure; it may include more than one chromosome type and will include debris. To determine the purity of a sort, i.e., the percentage of the sorted material that is of the actual chromosomal type desired, two methods of mathematical analysis have been developed. In the more general method, the bivariate data within an analysis region that includes the sort region, are fit with a series of bivariate Gaussian functions, one for each peak. In a simplified method, the data within the analysis region are transformed into a univariate distribution of either chromomycin A3 or Hoechst-33258 fluorescence. The peaks in these univariate distributions are fit with univariate Gaussian functions. In both methods the purity is determined mathematically. The results of both methods agree well with independent methods of analysis.

Flow cytometry measurements of human chromosome kinetochore labeling.

A method for the preparation and measurement of immunofluorescent human chromosome centromeres in suspension is described using CREST antibodies, which bind to the centromeric region of chromosomes. Fluorescein isothiocyanate (FITC)-conjugated antihuman antibodies provide the fluorescent label. Labeled chromosomes are examined on microscope slides and by flow cytometry. In both cases a dye which binds to DNA is added to provide identification of the chromosome groups. Sera from different CREST patients vary in their ability to bind to chromosome arms in addition to the centromeric region. Flow cytometry and microfluorimetry measurements have shown that with a given CREST serum the differences in kinetochore fluorescence between chromosomes are only minor. Flow cytometry experiments to relate the number of dicentric chromosomes, induced by in vitro radiation of peripheral blood cells to the slightly increased number of chromosomes with above-average kinetochore fluorescence did not produce decisive radiation dosimetry results.

[Clinical significance of chromosome number deviations in myelodysplastic syndromes and in acute non-lymphoid leukemia]. / A kromoszóma eltérések klinikai jelentösége myelodysplasiás szindrómában és akut nemlymphoid laukaemiában.

The cytogenetic data of 77 patients (47 adults and 30 children) with myelodysplastic syndromes and acute non-lymphoid leukemia are evaluated with regard to the morphological types of leukemia and prognosis. The groups of the adult patients were found to be different in the frequency and types of non-random chromosome aberrations. In patients with secondary leukemias and mutagen-related leukemias the incidence of chromosomal abnormalities was higher than in those with the idiopathic form of the disease. Specific abnormalities were total or partial loss of chromosome 5 and/or 7, and an additional chromosome 8. In contradistinction to these the patients with primary leukemias had specific translocations associated with pseudodiploidy. We found the frequency of aberrations in adults exposed to mutagen agents similar to that in children with ANLL, but the types of aberrations were similar to those of adults without any exposition. Comparing the median duration of remission and survival of patients' groups with different cytogenetic findings we found the chromosome aberrations to be of prognostic value. The present data demonstrate the usefullness of cytogenetic investigations in the diagnosis of the disease and in morphological and etiological classification of patients.

Synchronization of amniotic fluid cells for high resolution cytogenetics.

A high resolution technique was applied to amniotic fluid cells by synchronization. After inoculation, the cells were incubated for 30 h in the presence of either thymidine or 5-bromodeoxyuridine (BrdU). After removal of the blocking agent and addition of a low concentration of thymidine, the cells were incubated for another 6 1/2 7 h, then harvested in prometaphase without colcemid. This technique gives a mitotic index of 3.7 per cent after thymidine synchronization and of 3.2 per cent after BrdU synchronization, and more than half of the mitoses were in the earlier phases with the chromosomes showing more than 550 bands per haploid set. GBG, GTG, and RHG prometaphases are presented. Precise high-resolution banding of chromosomes of amniotic fluid cells can increase diagnostic accuracy.