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V Goyanes

Publications and source records attributed to V Goyanes.

At least 19 recordsLinked to original sources
PubMed2005

Architecture and evolution of dinoflagellate chromosomes: an enigmatic origin.

Dinoflagellates are a highly diversified group of unicellular protists that present fascinating nuclear features which have intrigued researchers for many years. As examples, a dense nuclear matrix accommodates permanently condensed chromosomes that are composed of fibers organized without histones and nucleosomes in stacked rows of parallel nested arches. The macromolecular chromosome structure corresponds to cholesteric liquid crystals with a constant left-handed twist. RNA acts to maintain the chromosome structure. Whole mounted chromosomes have a left-handed screw-like configuration with coils which progressively increase their pitch. This helical arrangement seems to be the result of a couple of narrow strands coiling together. Chromosomes do not show Q, G and C banding patterns. However, a roughly spherical differentiated upper end (primitive kinetochore?) and two differentiated coiling regions, the upper one composed of two to three coils where a couple of sister strands run together and parallel to each other, and the lower one where sister strands run out of phase by 180 degrees angular difference along the immediate next turns, can be distinguished. The chromosome segregation into two daughter chromatids begins at the telomere that attaches to the nuclear envelope, follows along the chromosome axis constituting first a Y-shaped and afterwards a V-shaped chromosome, which packs the newly synthesized DNA inside the "old" chromosome. Dividing chromosomes remain highly condensed, and the diameters of the new chromatids and the undivided chromosome are similar, but the number of arches is twice as large in G1 as in G2. The nuclear envelope remains through the cell cycle and shows spindle fibers, which penetrate intranuclear cytoplasmic channels during mitosis constituting an extra nuclear spindle. These and other cytogenetic features suggest that dinoflagellates are a group of enigmatic protists, unique and different from the usual eukaryotes. In contrast, DNA sequence studies propose that dinoflagellates are true eukaryotes, closely related to Apicomplexa, and ciliates (Alveolata), suggesting that the unusual features of chromosome and nuclear organization are not primitive but derived characters. Nevertheless, dinoflagellates have reached enigmatic specific nuclear and chromosome solutions, extremely far from those of other living beings.

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↗
PubMed2002

[Anterior sacral meningocele with Currarino's syndrome: report of two cases].

The anterior sacral meningocele is the congenital hernia of the thecal sac into the pelvis through a bone defect of the anterior wall of the sacrum. The association of anorectal anomaly, anterior sacral defect and presacral mass, known as Currarino's triad, can be diagnosed during the first decade of life in the eighty per cent of the cases However, in the incomplete syndrome, the symptoms appear in adults. In order to avoid late complications and due to the clear genetic origin of this uncommon pathology, the investigation of the family is of paramount importance for the early diagnosis and treatment. We review the literature and add two cases of anterior sacral meningocele with rectal fistula and incomplete Currarino's syndrome. In one of the cases the pathology is also present in another member of the same family and is complicated with meningitis. The importance of early diagnosis and treatment of these malformations and the need for fammily genetic investigation are discussed.

Adult↗
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↗
PubMed1997

AluI in situ digestion of human alphoid and classical satellite DNA regions: high-resolution digital image analysis of FISH signals from condensed and extended chromatin.

Human lymphocyte chromatin either extended or condensed in interphase nuclei and chromosomes was in situ digested by the restriction endonuclease AluI and then hybridized with alphoid probes specific for chromosome 1 (D1Z5 locus), for chromosome X (DXZ1 locus), and with a classical satellite DNA probe specific for chromosome 9 (D9Z1 locus). Fluorescent hybridization signals were quantified by digital analysis of high-resolution images obtained by a Photo-CD system in an attempt to analyze the differential DNA removal produced by AluI in specific repetitive DNA sequences with known restriction site frequency and distribution. The analysis of area and average pixel grey count of hybridization signals suggests that the greater the degree of chromatin stretching, the higher the accessibility of the probe and/or reporter molecules to the target. Nevertheless, this greater hybridization efficiency does not result in a higher fluorescence intensity due to dispersion of individual signals. Specific repetitive DNA at D9Z1 locus (classical) remained impervious to digestion, while that at DXZ1 (alphoid) was extensively removed, according to the frequency and distribution of restriction sites. Nevertheless, though the restriction sites were at least as frequent as at the DXZ1 locus, DNA at the D1Z5 locus (alphoid) was only partially removed. This indicates that chromatin organization within the C-band partially prevents extraction of alphoid sequences, supporting the hypothesis that alphoid DNA sequences are differentially organized among chromosomes. Overall, the same results were obtained from condensed and extended chromatin, suggesting that higher-order chromatin organization does not influence the in situ DNA cleavage and removal by AluI.

Cells, Cultured↗
PubMed1996

Quantification of C-ERB-B2 gene amplification in breast cancer cells using fluorescence in situ hybridization and digital image analysis.

Fluorescence in situ hybridization (FISH) allows detection of the intercellular heterogeneity of C-ERB-B2 gene amplification in uncultured breast cancer cells. Nevertheless, because high levels of amplification result in coalescence of signals, direct microscopy quantification is restricted to cells wih low levels of amplification or with dispersed signals. A methodology of digital image analysis, using surface and grey-level FISH signals as parameters that permit a rapid, objective, and accurate estimation of gene copy number, is presented. This procedure is independent of the signal overlapping and results in a more accurate quantification and characterization of tumor cell heterogeneity.

Breast Neoplasms↗
PubMed1996

Differential sensitivity of some human alphoid and classical satellite DNA regions from lymphocyte chromosomes to in situ exonuclease III digestion.

Fluorescent in situ hybridization of alphoid and classical satellite III DNA sequences was performed on fixed chromosomes from human lymphocytes that were previously digested in situ with exonuclease III to produce single-stranded DNA motifs. Digital image analysis showed that while labeled alphoid satellite DNAs produced signals of similar strength to thermally denatured chromosomes, those of classical satellite III DNAs of chromosomes 9 and Yq were around 50% weaker. This result shows a differential sensitivity of these satellite DNA regions to in situ exonuclease III digestion and suggests structural differences in the higher-order organization of both subchromosomal constitutive heterochromatic regions.

Chromosomes, Human↗
PubMed1995

High frequency of mutagen-induced chromatid exchanges at interstitial telomere-like DNA sequence blocks of Chinese hamster cells.

Interstitial telomere-like DNA sequence arrays of Chinese hamster Don cells were delineated by fluorescence in situ hybridization in quadriradial and triradial chromosome configurations induced by X-rays, mitomycin C and teniposide (VM-26). Around 40% of the scored exchanges involved a telomeric-like block of sequences at a rearrangement site. This frequency was independent of the DNA-damaging agent, and the result suggests a general recombinogenic capacity of interstitial telomere-like DNA sequence repeats that, at least in the case of the agents employed, seems not to be related to the initial mechanism of DNA damage.

Animals↗
PubMed1995

Long term effects of ritodrine on blood pressure and heart rate of adolescents exposed during the prenatal stage.

OBJECTIVE: To determine the long term cardiovascular effects in adolescents exposed to ritodrine during prenatal stage. STUDY DESIGN: Twenty-seven ritodrine-exposed subjects and 19 controls, of both sexes, clinically healthy, and born at normal term, were evaluated for systolic blood pressure (SBP), diastolic blood pressure (DBP) and heart rate (HR) each during 48 hours, with a Colin Ambulatory monitor. Data analysis were performed by individual Cosinor method, and non-parametric Mann Whitney's test and Lamotte procedures. RESULTS: Adolescents who were exposed to ritodrine showed losses in the HR rhythm and rising middle pulse values. Although SBP and DBP rhythm remains constant, the increase of maximum and minimum values for SBP as well as a rise of maximal rates of DBP suggest a possible hypertensive effect mediated by beta mimetic exposure. CONCLUSION: This study suggests a hypertensive effect mediated by beta mimetic exposure.

Adolescent↗
PubMed1995

X-ray biological dosimetry performed by selective painting of human chromosomes 1 and 2.

Fluorescence in situ hybridization with a cocktail of whole-chromosome probes, specific for human chromosomes 1 and 2, was performed to analyze the induction of translocations and dicentrics in human peripheral blood lymphocytes exposed to 100 KVp X-rays and cultured for 48 h. The dose-response curves for both aberration types were linear-quadratic, with similar coefficients, and after correction for genome content were a little lower than the dose-response curve for dicentrics established by conventional analysis. Three doses were also assessed after 76-h culture. The dicentric yield at 76 h was reduced compared with that at 48 h because of the existence of second and third division metaphases. The translocation yield did not reduce except at 4 Gy. The frequency of cells containing both dicentrics and translocations suggested that their occurrence is not correlated. The importance of stating the scoring criteria and of specifying the different types of aberrations is stressed.

Cells, Cultured↗
PubMed1995

Difference in constitutive heterochromatin behaviour between human amniocytes and lymphocytes detected by a sequential in situ exonuclease III digestion-random primer extension procedure.

Fixed chromosomes from human amniotic fluid cells and peripheral blood lymphocytes were digested in situ with exonuclease III and the single stranded DNA obtained was used as template for an in situ random primer extension. Under these conditions an R banding pattern, more evident in lymphocytes than in amniocytes, was obtained. Nevertheless, constitutive heterochromatin of chromosomes 1, 16, Yq, and mainly the pericentromeric region of chromosome 9 was far more intensely labelled in amniocytes than in lymphocytes. Fluorescence in situ hybridisation with a specific classical satellite DNA probe, showed that this differential labelling was dependent on a greater sensitivity of chromosome 9 constitutive heterochromatin to exonuclease III digestion in amniocytes than in lymphocytes, thus indicating qualitative differences in this region between both human cellular materials.

Adolescent↗
PubMed1995

Specific induction of uncoiling in NORs of human acrocentric chromosomes by 5-azacytidine and 5-azadeoxicytidine.

The organization of rDNA-containing chromatin was analysed by transmission electron microscopy after treatment of cultured human lymphocytes with 5-azacytidine (ACR) or 5-azadeoxicytidine (AdCR). The number of observed acrocentric chromosomes with satellites was significantly increased after treatment with low doses of ACR or AdCR during the last 24 h of culture, whereas with exposures during the last 7 h the number remained normal. The results suggest that the incorporation of ACR and AdCR in the early period of the S-phase may have reverted the non-satellized to satellized chromosomes. The cytidine analogues may have become more visible during secondary constriction thus changing the NOR structure leading to an increased number of satellized chromosomes.

Adult↗
PubMed1994

5-azacytidine produces differential undercondensation of alpha, beta and classical human satellite DNAs.

Fluorescence in situ hybridization employing human alphoid, beta and classical satellite DNA probes was performed on 5-azacytidine treated and untreated chromosomes obtained from human lymphocytes. The individual used in this study presented a polymorphism of constitutive heterochromatin of chromosomes 1 and 9 as revealed by in situ digestion with the restriction endonuclease Alul. Neither the alphoid nor the beta satellite DNA domains were susceptible to condensation-inhibition by 5-azacytidine. Only the classical satellite localized on chromosome 9 was affected. The constitutive heterochromatin size polymorphism was shown to depend mainly on variations of the classical satellite DNA domain. Therefore, condensation-inhibition, as a phenomenon which may modify the natural folding of the chromatin fibre, regionally affects human constitutive heterochromatin and seems to be dependent on the heterochromatic family.

Azacitidine↗