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J Gosálvez

Publications and source records attributed to J Gosálvez.

At least 19 recordsLinked to original sources
PubMed2006

Sperm DNA fragmentation in a random sample of the Spanish boar livestock.

A collection of 180 chilled boar semen samples, randomly chosen from stocks currently used for routine characterization of standard seminal quality, were studied for DNA fragmentation status using the sperm chromatin dispersion test and the DNA fragmentation index (DFI: percent of abnormal cell versus normal cells for DNA fragmentation) was determined. Values for sperm motility, acrosome status, coiled tails and abnormal head morphology, including presence and position of cytoplasmic droplets were also obtained. The DFI in the whole sample presented a wide range of variation with values oscillating between practically 0% and 47.95% and do not fit to a normal distribution. The most frequent classes (83.3%) presented a DFI lower than a 5%. Significant correlations between sperm DNA fragmentation and sperm motility, acrosome status, frequency of distal droplets, coiled tails and abnormal head morphology, were not observed. However, the presence of proximal cytoplasmic droplets showed a significant correlation with the level of DNA fragmentation observed in the ejaculated spermatozoa.

Acrosome↗
PubMed2004

Telomeric and interstitial telomeric-like DNA sequences in Orthoptera genomes.

A (TTAGG)n-specific telomeric DNA probe was hybridized to 11 orthopteroid insect genomes by fluorescence in situ hybridization. Nine different genera, mainly distributed within two evolutionary branches with male chromosome numbers 2n = 23 and 2n = 17 were included in the analysis. Telomere sequences yielded positive signals in every telomere and there was a considerable number of interstitial telomeric-like sequences, mainly located at the distal end of some, but not all, subterminal chromosome regions. One of the species, Pyrgomorpha conica, showed massive hybridization signals associated with constitutive heterochromatin. The results are discussed along two lines: (i) the chromosomal evolutionary trends within this group of insects and (ii) the putative role that ITs may play in a genome when they are considered telomere-derived, but not telomere-functional, DNA sequences.

Animals↗
PubMed2004

Spatio-temporal dynamics of a neutralized B chromosome in the grasshopper Eyprepocnemis plorans.

Spatial and temporal patterns of frequency variation for a neutralized B chromosome in the grasshopper Eyprepocnemis plorans were analyzed along six transects in the east of Spain to explore possible factors affecting the population dynamics of this polymorphism. Three parameters were employed to quantify B frequency: prevalence, load and mean frequency. Of them, load seemed to be the less sensitive parameter, probably due to its small range of variation. Prevalence, however, shows ample variation, but the mean frequency of B chromosomes per individual is the best parameter to characterize B frequency. Only river transects revealed significant differences among populations, and the use of two geographic explicit approaches (Mantel test and distograms) revealed significant isolation by distance (IBD), especially at the Segura River mouth, presumably due to low gene flow and drift. No temporal trend was found in the Segura River transects, which is consistent with the slow changes in B frequency expected during the random walk for neutralized B chromosomes. But these transects showed a clear spatial pattern, with B1 showing lower frequency in the upper course of this river. The present results provide the first empirical evidence of IBD in the evolution of a neutralized B chromosome, and support the notion that B dynamics at this evolutionary stage is best explained by a metapopulation approach.

Animals↗
PubMed2002

FISHing in the microwave: the easy way to preserve proteins. I. Colocalization of DNA probes and surface antigens in human leukocytes.

Most of the techniques for simultaneous visualization of proteic cell components and DNA probes are difficult to carry out. We have developed an alternative protocol for simultaneous visualization of DNA probes and cell surface antigens in human cells. The method exploits microwave energy to denature DNA and to anneal the probe after antigen fluorescent detection of unfixed cells previously embedded in a microgel. CD3 antigens and whole genome DNA probes or specific repetitive DNA sequences were colocalized in peripheral blood samples. The results show a strong, specific and consistent hybridization pattern in each cell that allowed correlation between cell subtype (PROTEINS) and nuclear phenotype (DNA).

CD3 Complex↗
PubMed2001

High frequency of constitutive alkali-labile sites in mouse major satellite DNA, detected by DNA breakage detection-fluorescence in situ hybridization.

DNA breakage detection-fluorescence in situ hybridization (DBD-FISH) is a new procedure for detecting and quantifying DNA breaks and alkali-labile sites in single cells. Cells trapped within an agarose matrix are deproteinized and treated with an alkaline unwinding solution that transforms DNA breaks and alkali-labile sites into single-strand DNA (ssDNA) motifs starting from the end of the break. These ssDNA motifs are susceptible to being hybridized with whole genome or specific DNA probes, and detected using current FISH procedures. As DNA breaks increase in a target region, more ssDNA is produced and more DNA probe hybridizes, thus increasing the FISH signal, which may be captured and analyzed using a digital image analysis system. This increase can be reflected in the surface area, mean and whole fluorescence intensity of the signal. When intact mouse splenocytes were processed with this technique using a whole genome probe, a very strong background signal was evident when compared with human blood leukocytes. In fact, when using 0.03M NaOH as the alkaline unwinding solution at 22 degrees C for 2.5min, the whole fluorescence intensity from mice cells was 50 times higher than that from human cells, thus suggesting the existence of a high frequency of constitutive alkali-labile sites in the DNA from mouse cells. Furthermore, when alkaline unwound mouse cells were simultaneously hybridized with the whole genome probe (FITC-revealed, green) and a major satellite DNA probe (Cy-3-labeled, red) both signals appeared co-localized. This result demonstrates that the high frequency of constitutive alkali-labile sites detected in the mouse genome is mainly located in the major satellite DNA sequences, resembling the findings from human 5bp classical satellite DNA sequences.

Alkalies↗
PubMed2001

DBD-fish on neutral comets: simultaneous analysis of DNA single- and double-strand breaks in individual cells.

Humanblood leukocytes exposed to X-rays were immersed in an agarose microgel on a slide, extensively deproteinized, and electrophoresed under neutral conditions. Following this single-cell gel electrophoresis assay, characteristics of DNA migration (i.e., area of the comet) are related to the DNA double-strand breaks (dsbs) yield. After electrophoresis, comets were briefly incubated in an alkaline unwinding solution, transforming DNA breaks and alkali-labile sites into restricted single-stranded DNA (ssDNA) motifs. These motifs behave as target sites for hybridization with a whole genome probe, following the DNA breakage detection-fluorescence in situ hybridization (DBD-FISH) procedure. As DNA breakage increases with dose, more ssDNA is produced in the comet by the alkali and more DNA probe hybridizes, resulting in an increase in the mean fluorescence intensity. Since radiation-induced DNA single-strand breaks (ssbs) are far more frequent than dsbs, the mean fluorescence intensity of the DBD-FISH signal from the comet is related to the ssb level, whereas the surface area of the same comet signal is indicative of the dsb yield. Thus, both DNA break types may be simultaneously analyzed in the same cell. This was confirmed in a repair assay performing the DBD-FISH on neutral comets from a human cell line defective in the repair of dsbs. Otherwise, treatment with hydrogen peroxide, a main inducer of ssbs, increased the mean fluorescence intensity, but not the surface, of X-ray-exposed human leukocytes.

Comet Assay↗
PubMed2001

Evidence of abundant constitutive alkali-labile sites in human 5 bp classical satellite DNA loci by DBD-FISH.

Human blood leukocytes within an agarose matrix were deproteinized and exposed to an alkaline denaturation that generates single-stranded DNA (ssDNA) starting from the ends of spontaneous basal DNA breaks and alkali-labile sites. Since the amount of ssDNA produced within a specific sequence area may be detected by hybridization with a specific probe, we quantified this in situ in different satellite DNA loci (DBD-FISH: DNA Breakage Detection FISH). The DBD-FISH signal, corrected for the respective FISH signals in metaphase, was remarkably strong in the 5bp classical satellite DNA domains analyzed (D1Z1, D9Z3, DYZ1), intermediate in the classical satellite 1 DNA sequences, and low in the alphoid satellite regions (D1Z5, DXZ1, all centromeres). This result is evidence of a high density of constitutive alkali-labile sites, probably abasic sites, within the 5bp satellite DNA sequences in human blood leukocytes. The presence and relative abundance of alkali-labile sites could explain the high frequency of spontaneous breakage and rearrangements in pericentromeric heterochromatin of chromosomes 1, 9, and 16, but not in Yqh, when this chromatin is undercondensed through spontaneous or induced demethylation, i.e. ICF syndrome or 5-azacytidine treatment.

Centromere↗
PubMed2001

Sau3A in situ digestion of human chromosome 3 pericentrometric heterochromatin. I. Differential digestion of alpha-satellite and satellite 1 DNA sequences.

In situ digestion with the restriction endonuclease (RE) Sau3A (Sau3A REISD) uncovers a polymorphism for the pericentromeric heterochromatin of human chromosome 3, which can be positively stained (3+) or not (3-), and has proven useful to differentiate donor and recipient cells after sex-matched bone marrow transplantation and to analyze the so-called hemopoietic chimerism. The aim of the present investigation was to obtain insight into the molecular basis of such polymorphism to optimize its use for chimerism quantification using methodological approaches other than REISD. To this end, fluorescence in situ hybridization (FISH) assays using probes for the satellite DNA sequences that mainly constitute chromosome 3 pericentromeric heterochromatin (alpha-satellite and satellite 1 DNA) were performed on control and Sau3A-digested chromosomes. The results obtained suggest that chromosome 3 alpha-satellite DNA is digested in all individuals studied, irrespective of the karyotype obtained by Sau3A REISD (3++, 3+-, 3--), and thus it does not seem to be involved in the polymorphism uncovered by Sau3A on this chromosome. Satellite 1 DNA is not digested in any case, and shows a polymorphism for its domain size, which correlates with the polymorphism uncovered by Sau3A in such a way that 3+ chromosomes show a large domain (3L) and 3- chromosomes show a small domain (3S). It seems, therefore, that the cause of the polymorphism uncovered by Sau3A on the pericentromeric region of chromosome 3 is a difference in the size of the satellite 1 DNA domain. Small satellite 1 DNA domains fall under the resolution level of REISD technique and are identified as 3-.

Centromere↗
PubMed2000

DNA breakage detection-FISH (DBD-FISH) in human spermatozoa: technical variants evidence different structural features.

Non-irradiated and X-irradiated (80 Gy) human spermatozoa were processed for in situ DNA breakage detection-FISH (DBD-FISH) of the whole genome, following two alternative variations of the basic technique. In the first, cells were initially incubated in the alkaline unwinding solution for transformation of DNA breaks into single-stranded DNA (ssDNA) to be hybridized, followed by the lysing solutions for protein removal. In the second, incubation in the lysing solutions was carried out before the denaturation step. The first approach yielded two subpopulations. While most sperm nuclei were faintly labeled and had chromocenters, a small subpopulation was strongly and homogeneously labeled, due to extensive DNA breakage. X-ray exposure increased the surface and mean fluorescence intensity. Otherwise, when the denaturation step was performed after protein extraction, all sperm nuclei yielded strong and dispersed FISH signals. Protein removal allows access of the unwinding solution to the DNA, which has abundant alkali-labile sites, and thus gives rise to large areas of ssDNA that are labeled by FISH. X-ray exposure increased the dispersion of FISH signals but decreased their mean fluorescence intensity. A linear dose-response was generated using the second experimental variant, being 30 Gy the lowest dose for detecting induction of damage by X-rays in mature sperm chromatin. These results indicate that DBD-FISH is not only useful for in situ detection of DNA breakage but also for revealing structural features of chromatin.

DNA↗
PubMed2000

DNA breakage detection-fish (DBD-FISH): effect of unwinding time.

DBD-FISH is a new procedure that allows detection and quantification of DNA breakage in situ within specific DNA target sites. Cells embedded in an agarose matrix on a slide are treated in an alkaline unwinding solution to transform DNA breaks into single-stranded DNA (ssDNA). After removal of proteins, DNA probes are hybridized and detected. DNA breaks increase the ssDNA and relax supercoiling of DNA loops, so more probe hybridizes, thereby increasing the surface area and fluorescence intensity of the FISH signal. The probe selects the chromatin area to be analysed. In order to restrict the extension of unwound ssDNA to a region closer to the origin of the DNA break, human leukocytes were processed for DBD-FISH with a whole genome probe, after a 10 Gy dose of X-rays, for various unwinding times: 5, 2 min and 30s. Two cell populations were detected after 30s, but not with the 5 or 2 min unwinding times. One cell group had small to medium haloes corresponding to the relaxation of DNA supercoiling after DAPI staining, and strong DBD-FISH labelling of induced DNA breaks, whereas the other cell group showed big haloes of DNA loop unfolding and an absence of DBD-FISH labelling. The latter group was similar to cells processed by DBD-FISH without the unwinding step. Thus, they should correspond to cells unaffected by the alkaline unwinding solution, possibly because very brief unwinding times do not allow the diffusion of the alkali into the cells deep within the gel, thus biasing the results. Taking this into account, 2 min seems to be the minimum unwinding time required for an accurate detection of a signal by DBD-FISH.

DNA↗
PubMed2000

A PCR product derived from female DNA with regional localization on the Y chromosome.

A 154-bp PCR product amplified from human female DNA mapped onto the Y chromosome under high-stringency in situ hybridization conditions. The female DNA sequence revealed an 89% homology with the HSDYZ1 sequence. When the same primers were used to amplify male DNA, a 154-bp DNA fragment was also obtained, showing a 98% homology with HSDYZ1. However, although the HSDYZ1 sequence is widely distributed along the long arm of the Y chromosome, both of these particular PCR products are di-regionally localized within this distal block of constitutive heterochromatin. In situ hybridization under lower stringency showed that these 154-bp sequences map both onto the autosomes and the Y chromosome. Overall, this paper shows (i) a new class of DNA sequences shared by the autosomes and the Y chromosome; and (ii) a substructured organization of some DNA repeats within the DYZ1 family that forms a large part of the constitutive heterochromatin of the Y chromosome.

Chromosome Mapping↗
PubMed2000

Specific heterochromatic banding of metaphase chromosomes using nuclear yellow.

The bis-benzimidazole compound nuclear yellow (NY) belongs to the same chemical family as the DNA binding fluorochromes Hoechst 33258 and Hoechst 33342. Spectroscopic studies of NY alone and in the presence of calf thymus DNA show high DNA binding affinity and behavior similar to the Hoechst fluorochromes above. Mitotic metaphase chromosomes from Balb/c mice stained with NY show C-banding and weak G/Q-banding, both of them disappearing after distamycin A (DA) or methyl green (MG) counterstaining. The same staining of human metaphase chromosomes from lymphocyte cultures, however, reveal only faint G/Q-banding (NY) and a characteristic DA-DAPI-like banding (NY-DA, NY-MG). Image analysis of NY stained human chromosomes, confirms that NY is suitable for studying polymorphisms affecting size in the pericentromeric heterochromatin of pairs 1, 9 and 16, and shows significant enhancement of NY fluorescence induced by DA in DA-DAPI heterochromatin. Our spectroscopic and cytological results show that NY, either alone or counterstained with DA or MG, can be used for DNA cytochemistry and chromosome banding. Possible mechanisms for the banding patterns induced by NY are discussed.

Animals↗
PubMed2000

Fluorescence in situ hybridization evaluation of minimal residual disease on stem-cell harvests.

The usefulness of fluorescence in situ hybridization (FISH) analysis to detect minimal residual disease (MRD) in autologous bone marrow and peripheral blood stem-cell harvests has been tested in three patients with hematologic malignancies. Conventional cytogenetics and FISH were used to characterize the leukemic clones identifying the specific chromosomal abnormalities (monosomy 7 in a myelodysplastic patient and trisomy 8 in two acute myeloid leukemic patients). Such analysis was useful to monitor the MRD persistent after treating these patients with intensive chemotherapy. The myelodysplastic patient underwent eight peripheral blood-stem cell harvests in which FISH detected the persistence of monosomy 7 cells, precluding their use for autologous transplantation. This patient relapsed and died. In two acute myeloid leukemia patients who underwent an autologous marrow harvest, FISH did not show a significant proportion of trisomy 8 cells. Nevertheless, autologous transplantation was not performed, owing to an insufficient CD34 cell content in the harvests. One of these patients relapsed with the reappearance of trisomy 8 and died. The other patient, on the contrary, is alive in complete remission 3 years after the bone marrow harvest. The usefulness and applicability of MRD quantification in stem-cell harvests is discussed on the basis of the sensitivity of the methodology applied.

Adolescent↗
PubMed1999

Histological damage in chronic hepatitis C is not related to the extent of infection in the liver.

It has not been completely elucidated whether the liver injury induced by the hepatitis C virus (HCV) is due to direct cytopathic damage or to an immune-mediated response against HCV-infected hepatocytes. In this work, we have determined the percentage of HCV-infected hepatocytes, the histological activity index, and the viremia levels in chronically HCV-infected patients with different grades of liver injury to investigate any possible correlation between them. For that purpose, liver biopsies from 27 patients with HCV chronic hepatitis were analyzed by in situ hybridization. This technique revealed that the percentage of infected hepatocytes ranged from 0.04% to 83.6%. Regarding the viremia levels, HCV RNA concentration ranged from 1.8 x 10(3) to 1.4 x 10(6) genome copies/ml. A significant correlation (r = 0.54; P = 0.003) between the percentage of infected hepatocytes and the viremia levels was found. In contrast, no correlation was observed between the percentage of HCV-infected hepatocytes or the viremia levels and the histological activity index. In conclusion, we have shown that the HCV viremia reflects the extent of the infection in the liver and that the liver injury in chronic HCV infection is not directly related to either the number of infected hepatocytes or the serum HCV RNA concentration.

Biopsy↗
PubMed1999

Chimerism quantification after sex-matched BMT: how probable is it to find donor/recipient pairs with distinguishable cells?

Chimerism quantification (CQ) after sex-matched bone marrow transplantation (BMT) is based on the identification of autosomal differences distinguishable at the chromosomal level, such as variations within constitutive heterochromatin between the recipient and the donor. The probability of finding distinguishable recipient/donor pairs at the karyotypic level depends on the frequency of the chromosome variants or morphs in the population, on whether recipient and donor are related, and if so, their kinship relation. We have developed a population genetics-based method that allows the estimation of the percentage of post-BMT CQ expected to be informative using any autosomal polymorphic marker. This method has been developed for the most common transplant situations, such as sibling-matched recipient/donor pairs, haploidentical related (parental/filial) pairs, and unrelated pairs. The method developed was applied to a polymorphism of the pericentromeric region of chromosome 3. This polymorphism becomes evident after in situ digestion with the restriction endonuclease Sau3A, and can be successfully used for CQ. It has been estimated that approximately 59% of the cases of BMT from unrelated donors, 36% of those from sibling donors, and 42% from parental/filial donors, are expected to be distinguishable for post-BMT CQ using this approach.

Bone Marrow Transplantation↗
PubMed1999

Polymorphisms for the size of heterochromatic regions allow sex-independent quantification of post-BMT chimerism targeting metaphase and interphase cells.

BACKGROUND AND OBJECTIVE: Fully quantitative cytological techniques for the analysis of hemopoietic chimerism are very limited and largely restricted to sex-chromosome detection after sex-mismatched bone marrow transplants (BMTs). The aim of the present investigation was to assess the usefulness of autosomal polymorphisms for the size of heterochromatic regions in the identification of donor and recipient cells and therefore in the quantification of the hemopoietic chimerism after sex-matched BMT. DESIGN AND METHODS: Hemopoietic chimerism was followed up in 3 transplanted patients targeting a polymorphism for the size of the pericentromeric heterochromatin (PCH) of chromosome 9, uncovered by restriction endonuclease (RE) in situ digestion (REISD) with the RE Sau3A, to differentiate donor and recipient cells on conventional bone marrow chromosome preparations. RESULTS: The polymorphism for the size of the PCH of chromosome 9 allowed differentiation of donor and recipient cells targeting both metaphase and interphase nuclei. The misidentification error for the polymorphism for the size of HPC of chromosome 9 was estimated as 1% for metaphases and 6-11% for interphases. The 3 cases studied showed complete chimerism in the first post-BMT sample analyzed, which was maintained in 2 of them. One patient relapsed and showed transient mixed chimerism. One month later, this patient achieved a second complete remission, showing complete chimerism again. In this patient, who received a sex-mismatched BMT, chimerism was also quantified by sex-chromosome identification using established methods, such as conventional cytogenetics and FISH, and the results obtained were similar to those rendered by Sau3A-REISD. INTERPRETATION AND CONCLUSIONS: The polymorphism for the size of the PCH of chromosome 9 uncovered by Sau3A-REISD allows accurate quantification of the hemopoietic chimerism after sex-matched BMT.

Bone Marrow Transplantation↗