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At least 1,261 records · Page 70Linked to original sources
PubMed2004

Improved testing for CMT1A and HNPP using multiplex ligation-dependent probe amplification (MLPA) with rapid DNA preparations: comparison with the interphase FISH method.

Charcot-Marie-Tooth disease type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP) are the two most common peripheral neuropathies, with incidences of about 1 in 2,500. Several techniques can be used to detect the typical 1.5-Mb duplication or deletion associated with these respective conditions, but none combines simplicity with high sensitivity. MLPA is a new technique for measuring sequence dosage. We have assessed its performance for the detection of the specific 1.5-Mb duplication/deletion by prospectively testing 50 patients referred with differential diagnoses of CMT or HNPP. Probes were designed to evaluate the TEKT3, PMP22, and COX10 genes within the CMT1A/HNPP region. We have compared the results with our existing fluorescence in situ hybridization (FISH) assay, which was performed in parallel. There was concordance of results for 49 patients. Of note, one patient showed an intermediate multiplex ligation-dependent probe amplification (MLPA) result with an abnormal FISH result, which is consistent with mosaicism. The assay works equally well with either purified DNA or rapid DNA preparations made by direct cell lysis. The use of the latter significantly reduces the cost of the assay. MLPA is a sensitive, specific, robust, and cost-effective technique suitable for fast, high-throughput testing and offers distinct advantages over other testing methods.

Charcot-Marie-Tooth Disease↗
PubMed2007

Nanotopographical stimulation of mechanotransduction and changes in interphase centromere positioning.

We apply a recently developed method for controlling the spreading of cultured cells using electron beam lithography (EBL) to create polymethylmethacrylate (PMMA) substrata with repeating nanostructures. There are indications that the reduced cell spreading on these substrata, compared with planar PMMA, results from a reduced adhesivity since there are fewer adhesive structures and fewer of their associated stress fibres. The reduced cell spreading also results in a reduced nuclear area and a closer spacing of centrosomes within the nucleus, suggesting that the tension applied to the nucleus is reduced as would be expected from the reduction in stress fibres. In order to obtain further evidence for this, we have used specific inhibitors of components of the cytoskeleton and have found effects comparable with those induced by the new substrata. We have also obtained evidence that these subtrata result in downregulation of gene expression which suggests that this may be due to the changed tension on the nucleus: an intriguing possibility that merits further investigation.

Cell Adhesion↗
PubMed1991

Standardization of interphase Ag-NOR measurement by means of an automated image analysis system using lymphocytes as an internal control.

Using an automated image analysis system, we have developed a procedure for standardizing the measurement of silver-stained proteins of the nucleolar organizer regions (NORs) in cancer cells, irrespective of the fixative employed and the time of the staining reaction. We observed that the area of Ag-NOR proteins from the same tumour was smaller in samples fixed with formalin-containing solutions, buffered formalin, and Bouin liquid, compared with those fixed with absolute ethanol and 'methcarn' solution (1.77 +/- 0.26micron 2 and 2.36 +/- 0.35 micron2 versus 3.34 +/- 0.54 micron2 and 3.72 +/- 0.61 micron2). Increased values of the Ag-NOR area were also observed after lengthening the silver staining reaction. However, in both cases no difference was observed in the ratio between Ag-NOR area of cancer cells and that of the lymphocytes infiltrating the stroma. The value of the ratio, which is called the 'Ag-NOR index', was in fact very similar, for the same cancer, after employing different fixatives or staining times. The use of lymphocyte Ag-NOR area as an internal control for the standardization of Ag-NOR evaluation in cancer tissues was made possible by the fact that lymphocyte Ag-NOR area is almost constant in human tumours independent of sex and age.

Adult↗
PubMed1995

Numerical aberrations of chromosomes 1 and 7 in renal cell carcinomas as detected by interphase cytogenetics.

Alcohol-fixed single cell suspensions of 37 renal cell carcinomas (RCCs) were assessed by both flow cytometry (FCM) and the fluorescence in situ hybridization (FISH) technique, using chromosome 1- and chromosome 7-specific centromere DNA probes. DNA diploidy or near-diploidy was observed in 30 of the 37 RCCs and only 12 of these (near-)diploid tumours were disomic for both chromosomes 1 and 7. Numerical aberrations of chromosome 1 and/or chromosome 7 were present in 18 of the 30 (near-)diploid RCCs and five of these cases showed monosomy for chromosome 1 in more than 50 per cent of the tumour cells. A double target FISH, with a centromeric and a telomeric specific probe for 1p36, excluded misinterpretation on the basis of clustering of 1q12, and suggested a complete loss of chromosome 1. All these five (near-)diploid RCCs with monosomy for chromosome 1 were eosinophilic chromophilic cell carcinomas, according to the Thoenes classification of RCC. This observation is of special interest, because it was recently concluded from cytogenetic studies that the diagnosis of chromophilic renal cell carcinoma must be considered as obsolete. Monosomy for chromosome 1 seems to be a non-random numerical aberration of (near-)diploid eosinophilic chromophilic cell carcinomas, and a gain of one or more chromosomes 1 appeared to be a common phenomenon in RCCs, especially in the DNA aneuploid tumours. As these chromosomal abnormalities were not found in the earlier classical cytogenetic studies, we conclude that in situ hybridization techniques are required in addition to chromosome banding techniques to obtain a complete characterization of the chromosome imbalances in RCCs.

Carcinoma, Renal Cell↗
PubMed1992

Rapid prenatal diagnosis of trisomy 18 and triploidy in interphase nuclei of uncultured amniocytes by non-radioactive in situ hybridization.

Two biotinylated chromosome-specific DNA probes were used to quantify the number of chromosomes 18 and 1 in uncultured amniocytes. Thirty-three samples of uncultured amniocytes were hybridized with a chromosome 18-specific DNA probe. Uncultured cells from two of the 33 samples were also hybridized with a chromosome 1-specific probe. Thirty of the samples were disomic with respect to chromosome 18; two samples were trisomic with respect to chromosome 18, and one sample was trisomic with respect to chromosomes 1 and 18. The two cases of trisomy 18 and the single case of triploidy were identified on uncultured cells within 48-72 h after amniocentesis. They were found among five samples from pregnant women who had amniocentesis because of an ultrasonographically identified fetal malformation. A trisomic karyotype could be diagnosed with certainty in uncultured amniocytes because the majority of the responding nuclei exhibited three hybridization signals. In normal cells, the majority of nuclei exhibited two signals. In no cases was there discordance between the genotype as predicted by in situ hybridization and that determined by cytogenetic analysis.

Amniotic Fluid↗
PubMed1994

Tetrasomy 12p (Pallister-Killian syndrome): ultrasound indicators and confirmation by interphase fish.

Tetrasomy 12p (Pallister-Killian syndrome) is a mosaic aneuploidy syndrome in which the isochromosome is present in amniocytes with a much greater percentage than fetal lymphocytes. Two new cases identified by prenatal diagnosis are reported. Indications for prenatal diagnosis were advanced maternal age and fetal anomalies. The most consistent reported prenatal ultrasound findings for tetrasomy 12p include polyhydramnios with short femurs and a diaphragmatic hernia. Recognition of congenital malformation patterns prenatally may allow appropriate selection of tissue for chromosome analysis. Molecular cytogenetic analysis using fluorescence in situ hybridization was used retrospectively to confirm the presence of the isochromosome 12p in various formalin-fixed fetal tissues. The levels of mosaicism detected in fetal and placental tissues were lower than those detected prenatally.

Abnormalities, Multiple↗
PubMed1994

Prenatal aneuploidy detection in interphase cells by fluorescence in situ hybridization (FISH).

FISH is a quick, inexpensive, accurate, sensitive and relatively specific method for aneuploidy detection in samples of uncultured chorionic villus cells and amniotic fluid cells. FISH allows detection of the autosomal trisomies 13, 18 and 21 and X and Y abnormalities and any other chromosome abnormality for which a specific probe is available. The detection rate of these abnormalities is high in informative samples which have a concordance of > 99.5% with cytogenetic results. A relatively high number of abnormal cases are found in uninformative samples. However, such samples should be regarded as samples to be investigated further. Clinical experience with the use of FISH for prenatal diagnosis is now beyond 10,000 cases; a number of clinical protocols and smaller trials have also been carried out, resulting in 90% of attempted analyses giving informative results with a high detection rate and extraordinarily low false-positive and false-negative rates. Unsolved problems remain, such as occasional technical failures, admixtures of maternal blood and up to 20% uninformative scoring results, especially for abnormal specimens. FISH is at present used as an adjunct to classical cytogenetic analysis. However, this should not be interpreted as meaning that FISH could not be used as a methodology in its own right. If FISH were to be considered a diagnostic test then this might be the case, due to the risk of false-negative and false-positive results and the fact that FISH does not allow a diagnosis of certain structural abnormalities. If, on the other hand, FISH is considered a screening test, which means that in all abnormal (or indeterminate) cases, classical cytogenetic analysis would follow the abnormal screening test, the accuracy which is potentially higher than for other screening methods, for example in cases of trisomy 21, justifies FISH as a prenatal screening test in its own right.

Amniotic Fluid↗
PubMed1993

Formation of compact globular particles in interphase nuclei from rat liver under the effect of polyanions.

Treatment of the isolated nuclei with polyanions (PA) (heparin and dextran-sulfate) at the PA/DNA ratio of about 1 in the 0.15-0.5 M ammonium acetate solution leads to rearrangement of the chromatin structure and formation of compact globular particles (GP) 40-70 nm in diameter, bound by fibrils of variable thickness (2-10 nm). GP formation is accompanied by a loss of nucleosomal periodicity in DNA organization. However, no considerable loss of the DNA contents has been found. At the PA/DNA ratio of more than 2, destruction of the DNP-network and aggregation of GP in larger globular structures are observed. The same effect is observed under the nuclease treatment. Under this condition nuclei lose a considerable part of DNA, but retain histones supposedly as histone-PA aggregates. Therefore the joint effect of PA and salt on nuclei at certain conditions may lead to formation of artefact structures.

Animals↗
PubMed1998

Colocalization of emerin and lamins in interphase nuclei and changes during mitosis.

Emerin is a nuclear membrane protein which is affected by mutation in X-linked Emery-Dreifuss muscular dystrophy. We have previously suggested that emerin is a member of a family of type II integral membrane proteins which associate with the nuclear lamina and which include lamina-associated proteins and the lamin B receptor. We now show that emerin in COS cells is not restricted to the nuclear rim but is also found at intranuclear sites, where it colocalizes with nuclear lamins B1, B2 and A/C. During mitosis, emerin is dispersed throughout the cell and then participates in the reconstitution of membranes around the daughter nuclei. Although emerin and lamins do not remain colocalized during mitosis, they all show some association with the midbody of the mitotic spindle.

Animals↗
PubMed1995

Calcium-independent, meiotic spindle-dependent metaphase-to-interphase transition in phorbol ester-treated mouse eggs.

Mouse eggs, arrested at metaphase II of meiosis, form pronuclei as a result of fertilization or exposure to parthenogenetic agents, such as the phorbol ester phorbol myristate acetate (PMA). Exposure of eggs to the microtubule inhibitor colcemid caused the disappearance of the meiotic spindle and prevented the PMA-induced release from metaphase. However, colcemid- and PMA-treated eggs which lacked spindles formed nuclei when treated in addition with the protein synthesis inhibitor cycloheximide or the protein kinase inhibitor 6-dimethlyaminopurine. To prevent an increase in intracellular calcium concentration ([Ca2+]i), eggs were exposed to the cell permeant acetoxymethyl ester (AM) form of the calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid (BAPTA). Nuclei formed in 45% of eggs treated with BAPTA AM and PMA and in 90% of eggs treated with BAPTA AM, PMA, and cycloheximide, suggesting that an increase in [Ca2+]i is not necessary for the PMA-induced release from metaphase. The [Ca2+]i did not change in eggs which formed nuclei in response to PMA, providing additional evidence that PMA activates eggs without elevating the [Ca2+]i.

Animals↗
PubMed1998

Spatial distributions of early and late replicating chromatin in interphase chromosome territories.

The surface area of chromosome territories has been suggested as a preferred site for genes, specific RNAs, and accumulations of splicing factors. Here, we investigated the localization of sites of replication within individual chromosome territories. In vivo replication labeling with thymidine analogues IdUrd and CldUrd was combined with chromosome painting by fluorescent in situ hybridization on three-dimensionally preserved human fibroblast nuclei. Spatial distributions of replication labels over the chromosome territory, as well as the territory volume and shape, were determined by 3D image analysis. During late S-phase a previously observed shape difference between the active and inactive X-chromosome in female cells was maintained, while the volumes of the two territories did not differ significantly. Domains containing early or mid to late replicating chromatin were distributed throughout territories of chromome 8 and the active X. In the inactive X-chromosome early replicating chromatin was observed preferentially near the territory surface. Most important, we established that the process of replication takes place in foci throughout the entire chromosome territory volume, in early as well as in late S-phase. This demonstrates that activity of macromolecular enzyme complexes takes place throughout chromosome territories and is not confined to the territory surface as suggested previously.

Cells, Cultured↗
PubMed1999

Spatial organization of four hnRNP proteins in relation to sites of transcription, to nuclear speckles, and to each other in interphase nuclei and nuclear matrices of HeLa cells.

RNA polymerase II transcripts are complexed with heterogeneous nuclear ribonucleoprotein (hnRNP) proteins. These proteins are involved in several aspects of the maturation and transport of hnRNA. We performed a detailed study of the spatial distribution of four hnRNP proteins (hnRNP C, I, L, and U) in HeLa nuclei, using immunofluorescent labeling and confocal microscopy. Despite the fact that hnRNP proteins have been shown to coimmunoprecipitate, a hallmark of hnRNP proteins, we find that hnRNP C, I, and L have a spatial nuclear distribution that is not related to that of hnRNP U. We also examined the distribution of hnRNP proteins in relation to that of nascent transcripts. The four hnRNP proteins that we examined are not enriched at sites of RNA synthesis. Using antibodies against the nuclear poly(A)-binding protein (PAB II) we investigated the relationship between the distribution of hnRNP proteins and that of nuclear domains (nuclear speckles) that are enriched in splicing factors, poly(A)+RNA, and PAB II. We found that the four hnRNP proteins are not enriched in these domains. This indicates that the poly(A)+RNA, present in high concentration in speckles, is not complexed with these hnRNP proteins. This is in agreement with the notion that poly(A)+RNA in speckles is different from ordinary hnRNA. Previously, we have shown that hnRNP proteins are the major protein components of the fibrogranular internal nuclear matrix (K. A. Mattern et al. (1996) J. Cell. Biochem. 62, 275-289; K. A. Mattern et al. (1997) J. Cell. Biochem. 65, 42-52). We observed that in nuclear matrices the spatial distributions of the four hnRNP proteins, like that of nascent RNA and PAB II, are essentially the same as observed in intact nuclei. Moreover, also in nuclear matrix preparations, like in intact nuclei, nascent RNA and PAB II have spatial distributions that differ from those of hnRNP proteins. Our results are compatible with the notion that hnRNP proteins are able to form complexes of many different, probably overlapping, compositions.

Cell Nucleus↗
PubMed1999

Association of human SCF(SKP2) subunit p19(SKP1) with interphase centrosomes and mitotic spindle poles.

In Saccharomyces cerevisiae, the initiation of DNA replication and mitotic progression requires SKP1p function. SKP1p is an essential subunit of a newly identified class of E3 ubiquitin protein ligases, the SCF complexes, that catalyze ubiquitin-mediated proteolysis of key cell-cycle-regulatory proteins at distinct times in the cell cycle. SKP1p is also required for proper kinetochore assembly. Little is known about the corresponding human homolog, p19(SKP1), except that it is expressed throughout the cell cycle and that it too is a component of an S-phase-regulating SCF-E3 ligase complex. Here we show by immunofluorescence microscopy that p19(SKP1) localizes to the centrosomes. Centrosome association occurs throughout the mammalian cell cycle, including all stages of mitosis. These findings suggest that p19(SKP1) is a novel component of the centrosome and the mitotic spindle, which, in turn, implies a physiological role of this protein in the regulation of one or more aspects of the centrosome cycle.

Animals↗