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Use of multicolour chromosome painting to identify chromosomal rearrangements in human lymphocytes exposed to bleomycin: a comparison with conventional cytogenetic analysis of Giemsa-stained chromosomes.

Exchange aberrations induced by bleomycin were identified by multicolour fluorescence in situ hybridisation (FISH) with probes for chromosomes 1, 2, and 3. The frequency and distribution of aberration types were compared to conventional metaphase analysis of Giemsa-stained chromosomes from the same human lymphocyte cultures. The total percentage of exchanges detectable by painting three pairs of chromosomes with separate colours was calculated as 40%. Giemsa staining revealed predominantly asymmetric chromosome exchanges, which are expected to comprise 50% of the total induced exchanges. Genomic exchange frequencies were, therefore, determined by multiplying the observed frequencies from FISH analysis by 2.5 and the number of asymmetric exchanges identified in Giemsa-stained slides by 2.0. By these calculations, the genomic exchange frequency calculated from chromosome painting exceeded that estimated by Giemsa-staining. This difference was due to the identification by chromosome painting of a unique class of cells in which chromosomes had undergone complex exchanges (nonreciprocal exchanges involving multiple mutual sites). The percentage of cells exhibiting exchanges was similar for both methods.

Azure Stains↗

Matrix-assisted laser desorption/ionization Fourier transform mass spectrometric analysis of oxygenated triglycerides and phosphatidylcholines in egg tempera paint dosimeters used for environmental monitoring of museum display conditions.

Oxidative changes in triacylglycerols and diacylphosphatidylcholines in egg tempera paint strips are used for chemical dosimetry of the quality of the museum environment. High-resolution matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS) was used as a rapid method for the determination of the exact elemental composition of the alteration products from diacylphosphatidylcholines and triacylglycerols. Light exposure of the egg tempera paints yields oxygenated diacylphosphatidylcholines and triacylglycerols. In the latter multiple incorporation of oxygen was observed as a recurring mass difference of 15.995, the exact atomic mass of oxygen. Owing to the high resolution of the FTMS data (routinely 20 000 at m/z 1000 in broadband mode), oxidation products with different elemental compositions but identical nominal mass could be distinguished. Products of oxidative cleavage of triacylglycerols were observed in samples exposed for longer times. The relative intensities of the peaks of singly and multiply oxygenated triacylglycerols were used to derive the degree of oxygenation of the egg lipids in the tempera paint dosimeters. The degree of oxygenation was found to be directly related to the light exposure time. Exposure to elevated temperature (60 degrees C) for a period of 21 days did not lead to oxygenation of the triacylglycerols and diacylphosphatidylcholines. Exposure to NO(x) and SO(2) in the dark greatly increased the degree of oxygenation. Addition of lead- or copper-containing pigments to the egg binding medium (and subsequent storage for 6 months in the dark) led to accelerated conversion of egg lipids to oxidised products.

Journal Article↗

Separation techniques for the analysis of artists' acrylic emulsion paints.

Emulsion paints are complex chemical systems. The main problems in their characterization are related to the similarities in polymer composition and to the presence of many different types of additives. Thus high resolution separations, sensitivity, and response specificity are required to identify simultaneously the polymer matrix and the minor components. Especially surfactants and pigments are thought to affect significantly the properties of the paint layers during ageing and their identification is the first step in evaluating the behaviour of these products in working conditions. Representative samples of acrylic emulsion paints for artists have been investigated by pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS) and size exclusion chromatography-Fourier transform infrared spectroscopy (SEC-FTIR), and the results have been compared. All the analysed samples were bound in ethyl acrylate-methyl methacrylate, poly(EA-co-MMA), or n-butyl acrylate-methyl methacrylate, poly(nBA-co-MMA), copolymers. Two types of nonionic surfactants, ethoxylated fatty alcohols and alkylaryl polyethoxylates, commonly used as emulsifiers and dispersive agents have been identified, together with a number of organic pigments.

Journal Article↗

Homologies between human and marmoset (Callithrix jacchus) chromosomes revealed by comparative chromosome painting.

Regions of DNA homology between human and marmoset (Callithrix jacchus) chromosomes have been demonstrated using fluorescence in situ hybridization. All 24 chromosome paints and two centromere repeat sequences from Homo sapiens (HSA) have been annealed to previously G-banded metaphase spreads of Callithrix jacchus. All human paint probes, except Y, successfully hybridized to marmoset chromosomes. Fifteen of them hybridized to one region only, seven to two regions, and paint 1 to three regions. Homologies proposed from previous banding comparisons have been confirmed for HSA 2, 4-6, 10-12, 18, 19, 21 and X and partially confirmed for HSA 1 and 3, but were not in agreement for HSA 14 and 17. Human centromere repeat sequences for X and 18 did not hybridize to marmoset chromosomes. Because, at present, there is the confusion situation of several different numbering systems for marmoset chromosomes, we propose a new simpler nomenclature based on descending order of chromosome size.

Animals↗

Comparative chromosome painting in mammals: human and the Indian muntjac (Muntiacus muntjak vaginalis).

We have used human chromosome-specific painting probes for in situ hybridization on Indian muntjac (Muntiacus muntjak vaginalis, 2n = 6, 7) metaphase chromosomes to identify the homologous chromosome regions of the entire human chromosome set. Chromosome rearrangements that have been involved in the karyotype evolution of these two species belonging to different mammalian orders were reconstructed based on hybridization patterns. Although, compared to human chromosomes, the karyotype of the Indian muntjac seems to be highly rearranged, we could identify a limited number of highly conserved homologous chromosome regions for each of the human chromosome-specific probes. We identified 48 homologous autosomal chromosome segments, which is in the range of the numbers found in other artiodactyls and carnivores recently analyzed by chromosome painting. The results demonstrate that the reshuffling of the muntjac karyotype is mostly due to fusions of huge blocks of entire chromosomes. This is in accordance with previous chromosome painting analyses between various Muntjac species and contrasts the findings for some other mammals (e.g., gibbons, mice) that show exceptional chromosome reshuffling due to multiple reciprocal translocation events.

Animals↗

Delineation of marker chromosomes by reverse chromosome painting using only a small number of DOP-PCR amplified microdissected chromosomes.

A new procedure for determining the chromosomal origin of marker chromosomes has been carried out. The origin of marker chromosomes that were unidentifiable by standard banding techniques could be verified by reverse chromosome painting. This technique includes microdissection, followed by in vitro DNA amplification and fluorescence in situ hybridization (FISH). A number of marker chromosomes prepared from unbanded and from GTG-banded lymphocyte chromosomes were collected with microneedles and transferred to a collection drop. The chromosomal material was amplified by a degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR). The resulting PCR products were labelled by nick-translation with biotin-11-dUTP and used as probes for FISH. They were hybridized onto normal metaphase spreads in order to determine the precise regional chromosomal origin of the markers. Following this approach, we tested 2-14 marker chromosomes in order to determine how many are necessary for reverse chromosome painting. As few as two marker chromosomes provided sufficient material to paint the appropriate chromosome of origin, regardless of whether the marker contained heterochromatic or mainly euchromatic material. With this method, it was possible to identify two marker chromosomes of a healthy proband [karyotype: 48,XY,+mar1,+mar2] and an aberrant Y chromosome of a mentally retarded boy [karyotype: 46,X, der(Y)].

Adult↗

Detection of amplified DNA sequences by reverse chromosome painting using genomic tumor DNA as probe.

A modification of "reverse chromosome painting" was carried out using genomic DNA from tumor cells as a complex probe for chromosomal in situ suppression hybridization to normal metaphase chromosome spreads. Amplified DNA sequences contained in such probes showed specific signals, revealing the normal chromosome positions from which these sequences were derived. As a model system, genomic DNAs were analyzed from three tumor cell lines with amplification units including the proto-oncogene c-myc. The smallest amplification unit was about 90 kb and was present in 16-24 copies; the largest unit was bigger than 600 kb and was present in 16-32 copies. Specific signals that co-localized with a differently labeled c-myc probe on chromosome band 8q24 were obtained with genomic DNA from each cell line. In further experiments, genomic DNA derived from primary tumor material was used in the case of a male patient with glioblastoma multiforme (GBM). Southern blot analysis using an epidermal growth factor receptor gene (EGFR) probe that maps to 7p13 indicated the amplification of sequences from this gene. Using reverse chromosome painting, signals were found both on band 7p13 and bands 12q13-q15. Notably, the signal on 12q13-q15 was consistently stronger. The weaker 7p13 signal showed co-localization with the major signal of the differently labeled EGFR probe. A minor signal of this probe was seen on 12q13, suggesting cross-hybridization to ERB3 sequences homologous to EGFR. The results indicate co-amplification of sequences from bands 12q13-q15, in addition to sequences from band 7p13. Several oncogenes map to 12q13-q15 providing candidate genes for a tumor-associated proto-oncogene amplification. Although the nature of the amplified sequences needs to be clarified, this experiment demonstrates the potential of reverse chromosome painting with genomic tumor DNA for rapidly mapping the normal chromosomal localization of the DNA from which the amplified sequences were derived. In addition, a weaker staining of chromosomes 10 and X was consistently observed indicating that these chromosomes were present in only one copy in the GBM genome. This rapid approach can be used to analyze cases where no metaphase spreads from the tumor material are available. It does not require any preknowledge of amplified sequences and can be applied to screen large numbers of tumors.

Chromosome Aberrations↗

Detecting 'hidden' exchange events within X-ray-induced aberrations using multicolour chromosome paints.

In a recent series of papers we pointed out that any complex exchange (an interaction involving three or more breaks) can yield a wide range of aberration configurations. If these aberrations are detected with a single chromosome paint and total centromere probe, the majority will be scored as either 'simple' exchanges, apparently derived from two breaks, which we term pseudosimples, or as complexes which appear to have fewer breaks than were actually involved, which we term reduced complexes. In this study, metaphases from X-irradiated human fibroblasts were hybridized to two distinctly labelled composite probes (paints) for chromosomes 1 and 2. All centromeres were marked by differential 4',6-diamidino-2-phenylindole (DAPI) staining. Aberrations derived from exchanges involving both paints were characterized. With this method, we are able to identify some of the additional exchange events, so confirming the existence of these two classes of aberration. Ultimately we believe it can be used to estimate their frequency.

Cells, Cultured↗

Analysis of bulk and inorganic degradation products of stones, mortars and wall paintings by portable Raman microprobe spectroscopy.

This work reports the use of a portable Raman microprobe spectrometer for the analysis of bulk and decaying compounds in carbonaceous materials such as stones, mortars and wall paintings. The analysed stones include limestone, dolomite and carbonaceous sandstone, gypsum and calcium oxalate, both mono- and dihydrated, being the main inorganic degradation products detected. Mortars include bulk phases with pure gypsum, calcite and mixtures of both or with sand, soluble salts being the most important degradation products. The pigments detected in several wall paintings include Prussian blue, iron oxide red, iron oxide yellow, vermilion, carbon black and lead white. Three different decaying processes have been characterised in the mortars of the wall paintings: (a) a massive absorption of nitrates that reacted with calcium carbonate and promoted the unbinding of pigment grains, (b) the formation of black crusts in the vault of the presbytery and (c) the thermodecomposition of pigments due to a fire.

Journal Article↗

Raman spectroscopic study of a post-medieval wall painting in need of conservation.

Raman spectroscopic studies of four specimens from an important angel wall painting in need of conservation work in a medieval church have provided some information about the pigments and pigment compositions which will influence possible future preservation and restoration strategies. Excitation of the Raman spectra at 1,064 nm in macroscopic mode and at 785 nm in microscopic mode revealed that the white pigment on the angel's wings was a mixture of barytes with calcite and lead white in minor composition. Although the specimens provided were not directly associated with coloured regions of the painting, yellow and blue microcrystals were found and they were identified as chrome yellow and lazurite, respectively. Red and brown particles were identified as cinnabar/vermilion and haematite. Several green particles were also found but could not be identified. The green and blue crystals could be related to neighbouring coloured regions of the artwork and the yellow colour could be identified as a background to the angel figure. Particles of carbon were found to be dispersed throughout the specimens and can be ascribed to soot from candles, heating stoves or oil lamps providing lighting in the church. No evidence for biological deterioration was found from the spectra. The unusual pigment palette is strongly suggestive of a later date of painting than was originally believed but there is a possibility that an earlier rendition exists underneath. Following a review of the spectroscopic data, a more extensive sampling protocol is recommended, from which some stratigraphic evidence could identify the underlying plaster and possible artwork.

Journal Article↗

The de Brécy Madonna and Child tondo painting: a Raman spectroscopic analysis.

Raman spectra have been obtained from a Madonna and Child tondo painting, known as the de Brécy Tondo. Despite the provision of only a small number of microscopic samples, definitive spectra were obtained from mineral pigments. From one specimen, spectra of an organic binder enabled the consideration of several possibilities to be accomplished and a suggestion proposed for the medium. In another specimen the identification of the spectral signatures of Prussian blue, which was only synthesised some 200 years after the predicated date of execution of the painting, indicated that some unrecorded restoration had been undertaken later in the painting's history. Research carried out on this tondo from 1987 to 1991 indicated the probability that it is the work of Raphael, a conclusion supported by further research recently undertaken on the provenance. The stylistic similarity of the tondo to Raphael's Sistine Madonna is very clear; the pigments identified in this analysis are consistent with a Renaissance attribution for the de Brécy Tondo.

Journal Article↗

Homologous and heterologous FISH painting with PARM-PCR chromosome-specific probes in mammals.

Numerous loci can be amplified by PARM-PCR on 300 sorted chromosomes in low-stringency conditions (annealing at 30 degrees C during the two first cycles) to produce a probe that can be used in FISH painting experiments. We demonstrate that, depending on the primer chosen for the amplification, patterns of different quality can be obtained. In order to design a primer that allows amplification of coding sequences, we have shown that motifs of at least seven glutamic acid repeats (GAG or GAA codons) are present in human proteins more frequently than expected. Moreover, these repeats do not correspond to triplet expansion and can be conserved between species. Using probes prepared from sorted chromosomes with (GAG)7 primer, we were able to achieve homologous FISH painting on human, porcine, ovine, and bovine species, and bidirectional heterologous FISH painting between human and porcine species. As an example, using probes for human Chromosome (Chr) 19 and porcine Chrs 1 and 6, we clearly defined the regional homologies existing between those chromosomes.

Animals↗

Comparison of horse chromosome 3 with donkey and human chromosomes by cross-species painting and heterologous FISH mapping.

The melanocortin 1 receptor (MC1R), mast/stem cell growth factor receptor (KIT), and platelet-derived growth factor receptor alpha (PDGFRA) are loci that all belong to equine linkage group 2 (LG2). Of these, KIT was fluorescent in situ hybridization (FISH) mapped to ECA3q21 with equine cDNA and heterologous porcine BAC probes, while MC1R was localized to ECA3p12 and PDGFRA to ECA3q21 with heterologous porcine BAC probes. A three-step comparison between ECA3 and donkey chromosomes was carried out. First, microdissected ECA3 painting probe was used on donkey chromosomes, which showed disruption of the equine synteny. Next, human (HSA) Chromosomes (Chrs) 16q and 4 specific paints, known to be homologous to ECA3p and 3q, respectively, were applied to detect homologous chromosomal segment(s) in donkey. Finally, four genes (MC1R, ALB, PDGFRA, KIT) and two equine microsatellite markers (SGCV18 and SGCV33) located on ECA3 were FISH mapped to donkey chromosomes. The findings refined the cross species painting homology results and added six new markers to the nascent donkey gene map. The hypothesis that Tobiano coat color in horses may be associated with a chromosomal inversion involving genes within LG2 was tested by G-banding-based cytogenetic analysis and ordering of four loci-KIT, PDGFRA, albumin (ALB), and MC1R-in Tobiano and non-tobiano (homozygous as well as heterozygous) horses. However, no difference either in banding patterns or location/relative order of the genes was observed in the three classes. The study highlights successful FISH mapping of BAC probes across evolutionarily diverged species, viz., pig and horse/donkey, and represents the first use of large-sized individual clones across distantly related farm animals.

Animals↗

A novel source of highly specific chromosome painting probes for human karyotype analysis derived from primate homologues.

We have established a series of highly specific painting probes for human acrocentric chromosomes. These chromosomes are involved in the formation of the nucleolar organizer region (NOR) and show DNA sequence homologies within their pericentric heterochromatin. To date, these chromosomes have shown considerable cross hybridization in chromosome painting experiments. Our probe set has been established from primate homologues that are not involved in the NOR in that particular species or from species in which highly repetitive sequences have undergone rapid sequence divergence. The new painting probes should be of particular value for automated microscopy, for which highly specific signals are required as they are recorded at low magnification, e.g. when scoring chromosome 21 domains in interphase nuclei.

Animals↗

Injury incidence rates in a paint company on rotating production shifts.

Reports of work accidents and increased absence due to sickness among shift workers have suggested that their work performance and health are adversely affected by interference with the body's normal circadian or biological rhythm. Since the rotating shift is the most disruptive of the work regimens, an investigation was undertaken of the reportable occupational injuries of 1700 paint production and associated employees of one company working only on rotating shift. Their annual accident incidence rates averaged over 10 years were compared to the average annual rate reported by the Department of Labor for the entire United States paint manufacturing industry representing predominately day and non-rotating shift workers. The significance of the hour of the accident incidence rates was tested by chi-square statistics using loglinear models appropriate for null hypotheses that assume no effect due to shift work. The incidence rates of the company studied were lower than those of the U.S. paint industry, suggesting work safety practices uniquely related to the company studied may have predominated. Within the company, however, some time-of-day and circadian effects were indicated by an increased rate of accidents on the night shift, particularly during the last 3 h of the shift.

Accidents, Occupational↗

Diffuse pulmonary injury following paint remover exposure.

A case of paint remover inhalation causing pulmonary edema and pleural effusions is described. Methylene chloride, an ingredient of paint removers, can cause pulmonary edema and carbon monoxide poisoning. The expanding interest in home projects involving furniture refinishing and paint removal puts a large segment of the population at risk. The implications of such exposure need to be recognized by the medical community.

Adult↗

The persistence of aberrations in mice induced by gamma radiation as measured by chromosome painting.

Fluorescence in situ hybridization, or chromosome painting, has become an invaluable tool in the cytogenetic evaluation of historical or chronic exposure because it can be used to detect stable genetic damage, such as translocations, which persist through cell division, quickly and easily. The recent development of chromosome-specific composite DNA probes for the mouse has allowed the use of chromosome painting in this commonly used animal model. In order to measure the persistence of radiation-induced translocations, C57BL/6 female mice were given a whole body acute dose of 0, 1, 2, 3 or 4 Gy 137Cs gamma rays at 8 weeks of age. Metaphase chromosomes from both peripheral blood and bone marrow cells were obtained from four mice in each dose group at 1, 8, 15 and 30 days post-irradiation. Chromosomes 2 and 8 were painted, while the remaining chromosomes were counterstained with propidium iodide. DAPI counterstain was used to differentiate between translocations and dicentrics because it brightly labels the centromeric heterochromatin. The equivalent of 100 cells from each tissue was scored from each mouse. The results show that the percentage of reciprocal translocations, at least at doses of 3 Gy or lower, did not decrease with time in either tissue. In contrast, the frequency of non-reciprocal translocations induced by doses of 3 Gy or lower, remained unchanged in the peripheral blood, but decreased after a week in the bone marrow, then remained constant. An increase in these two types of aberration was observed between 15 and 30 days in the bone marrow and may have been due to clonal expansion. Dicentrics decreased with time in both tissues, almost none remained in the bone marrow after 8 days. These data suggest that reciprocal translocations are persistent and will serve as an effective biodosimeter for radiation exposure.

Animals↗

Partial triplication (7q) in a child with acute lymphoblastic leukemia demonstrated with conventional cytogenetics, PRINS, and chromosome painting.

In this report we describe a partial triplication (7q) and other structural aberrations found in a child with acute lymphoblastic leukemia (ALL), and we demonstrate the importance of PRimed IN Situ labeling (PRINS) and chromosome painting as a support to banding analysis for the clarification of complex structural chromosome rearrangements. Initially, the der(7) was interpreted as der(7)t(1;7;7). However, PRINS and chromosomes painting showed that der(7) consisted entirely of chromosome 7 material. Further, a derivative chromosome interpreted by banding analysis as a der(17)t(?1;17) was shown to be der(17)t(13;17) by the newly developed PRINS painting technique.

Child↗