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Direct detection of repetitive, whole chromosome paint and telomere DNA probes by immunogold electron microscopy.

Biotinylated repetitive, whole chromosome paint and telomere DNA probes were investigated at the electron microscope level after non-isotopic in situ hybridization and direct immunogold detection. The protocol described allowed the visualization of a biotinylated chromosome 1 specific satellite DNA probe in the light microscope without silver intensification. This sensitive method was exploited to analyse factors contributing to signal strength in immunogold chromosome painting. Furthermore, it allowed us to investigate the distribution of (TTAGGG)n telomere repeats in human metaphase chromosomes and interphase nuclei. Telomeric and internal (TTAGGG)n repeats were detected at high spatial resolution in human metaphase chromosomes. In the periphery of lymphocyte interphase nuclei, two types of telomere hybridization signals were observed. They differed remarkably in compactness, indicating two types of chromatin conformation present at the interphase telomeres in situ.

Base Sequence↗

Chromosome painting defines genomic rearrangements between red howler monkey subspecies.

We hybridized whole human chromosome-specific DNA libraries to chromosomes of two supposed subspecies of Alouatta seniculus: Alouatta seniculus sara and Alouatta seniculus arctoides. The number of hybridization signals per haploid set is 42 in A. s. sara and 43 in A. s. arctoidea; the two karyotypes differ by at least 16 chromosomal rearrangements, including numerous translocations. An unusual sex chromosome system is shared by both taxa. The sex chromosome system results from a Y translocation with a chromosome homologous to parts of human chromosome 3/15 and can be described as X1X2Y1Y2/X1X1X2X2 (male/female). Both red howlers also have microchromosomes, a highly unusual karyological trait not found in other higher primates. These microchromosomes are not hybridized by any human chromosome paint and therefore are probably composed of repetitive DNA. It is well known that New World monkeys have high karyological variability. It is probable that molecular cytogenetic analyses including chromosome painting will permit an accurate reconstruction of the phylogeny of these monkeys and help establish the ancestral karyotype for higher primates.

Alouatta↗

Interstitial deletion of chromosome 6q: precise definition of the breakpoints by microdissection, DNA amplification, and reverse painting.

Routine chromosomal analysis using GTG-banding alone showed a mosaic terminal deletion of 6q in a 14-week-old boy with developmental retardation, facial anomalies, agenesis of corpus callosum, cleft palate, hypotonia, short neck and pterygium colli, and minor anomalies of hands and feet. Discrepancies between the clinical findings on our patient and those described in the literature on patients having terminal deletions led to a more precise analysis of the karyotype. Reverse painting was performed on normal G-banded metaphases for exact determination of the breakpoints and on metaphases of the patient for evaluation of mosaicism. A DNA library that was obtained by microdissection of three deleted chromosomes 6 was used as a painting probe. Subsequent DNA amplification was performed with the help of topoisomerase-pretreated degenerate oligonucleotide primers. Unexpectedly, the hybridization pattern on normal metaphase chromosomes revealed an interstitial deletion with breakpoints at 6q25.1 and 6q27 instead of a terminal deletion. Hybridization on metaphases of the patient showed one deleted chromosome 6 in all metaphases analyzed at a higher resolution rather than mosaicism as previously assumed [karyotype, 46,XY,del(6)(q25.1 --> q27)]. We assume that in the single cases of 6q- described in the literature the deletions are misclassified. This might be due to difficulties in distinguishing between interstitial and terminal deletions at 6q and in precisely defining chromosomal breakpoints after GTG-banding alone.

Abnormalities, Multiple↗

The state of conservation of painted surfaces in the presence of accelerated ageing processes monitored by use of FT-Raman spectroscopy and multivariate control charts.

A new method has been developed for monitoring the degradation of paintings. Two inorganic pigments (ultramarine blue and red ochre) were blended with linseed oil and spread on canvas. Each canvas was subjected to simulated accelerated ageing in the presence of typical degradation agents (UV radiation and acidic solution). Periodically the painted surfaces were analysed by FT-Raman, to investigate the status of the surface. The data obtained were analysed by principal component analysis (PCA). Finally the Shewhart and cumulative sum control charts based on the relevant principal components (PC) and the so called scores monitoring and residuals tracking (SMART) charts were built. The method based on the use of PC to describe the process was found to enable identification of the presence of relevant modification occurring on the surface of the samples studied.

Journal Article↗

In situ analysis of mediaeval wall paintings: a challenge for mobile Raman spectroscopy.

Raman spectroscopy is an analytical technique, which is gaining attention as a molecular technique for the investigation of objects of art. Especially the non-destructive properties of the method make this application suitable for the in situ analysis of artefacts. However, although using mobile, fibre optics Raman instrumentation for this type of research seems to be straightforward, some practical obstacles may hamper the investigation. In this paper, pitfalls and solutions are described when applying a dedicated spectrometer to the analysis of mediaeval wall paintings. It is shown how some practical problems may be overcome, and the results of the analysis are presented. Although the mediaeval wall paintings from the chapel of the castle of Ponthoz are well-preserved, still some interesting degradation phenomena could be observed: the identification of a black degradation product, likely to be meta-cinnabar, a degradation product of the red pigment vermilion (HgS); the formation of gypsum (CaSO4.2H2O) as a weathering product of calcium carbonate (CaCO3); the observation of copper(II)hydroxychlorides.

Journal Article↗

Analytical study of canvas painting collection from the Basilica de la Virgen de los Desamparados using SEM/EDX, FT-IR, GC and electrochemical techniques.

A new method, based on the combined use of scanning electron microscopy-x-ray microanalysis, Fourier transform infrared spectroscopy, gas chromatography and differential pulse voltammetry, is proposed which permits a parallel investigation of organic and inorganic materials and the characterization of the artistic technique employed in canvas painting. The proposed method has led to analyze succesfully the canvas painting collection exhibited in the Basílica de la Virgen de los Desamparados (Valencia, Spain) which includes art works from 17th to 20th century.

Journal Article↗

Accumulation of lactate by supercooled hatchlings of the painted turtle (Chrysemys picta): implications for overwinter survival.

Hatchlings of the North American painted turtle (Chrysemys picta) typically spend their first winter of life inside the shallow, subterranean nest where they completed embryogenesis the preceding summer. Neonates at northern localities consequently may be exposed during winter to subzero temperatures and frozen soil. Hatchlings apparently survive exposure to such conditions by supercooling, but the physiological consequences of this adaptive strategy have not been examined. We measured lactate in hatchling painted turtles after exposure to each of three temperatures (0 degree C, -4 degrees C, and -8 degrees C) for three time periods (5 days, 15 days, and 25 days) to determine the extent to which overwintering hatchlings might rely on anaerobic metabolism to regenerate ATP. Whole-body lactate increased with increasing duration of exposure and decreasing temperature, and the highest levels were associated with the group that experienced the highest mortality. These results indicate that animals may develop a considerable lactic acidosis during a winter in which temperatures fall below 0 degree C for weeks or months and that accumulation of lactate may contribute to mortality of overwintering animals.

Acidosis, Lactic↗

A novel applicator for the selective painting of pre-retinal structures during vitreoretinal surgery.

PURPOSE: To demonstrate a novel applicator, the so-called vitreoretinal-internal limiting membrane colour enhancer to achieve the selective painting of pre-retinal structures. METHODS: The VINCE is a modified backflush needle, containing an adjustable silicone tube, which is surrounded by a metal cannula. The tube is connected to a dye container in the handpiece of the applicator. RESULTS: The instrument was successful in painting the retinal surface sufficiently and was found to be particularly useful to avoid unintended staining of the peripheral or foveal retina. The newly developed customized cartridge-loading system contains the ready prepared dye, avoiding time-consuming dilution and preparation of the vital dye during surgery in the OR. The VINCE is effective in restricting the dye only to the area of surgical interest. CONCLUSION: The VINCE is a useful tool to limit intraocular dye to the required area to minimize possible side effects to intraocular retinal structures.

Animals↗

On the frequency of chromosome exchanges in a control population measured by chromosome painting.

Chromosome painting has been shown to be a valid and rapid method for quantifying structural chromosome rearrangements in human lymphocytes. The method is particularly useful for detecting stable aberrations which are difficult and expensive to quantify with classical methods. The inherent stability of translocations has enabled them to be used as a biodosimeter for chronic and temporally displaced exposure to radiation. Translocations may also be useful for quantifying chronic exposure to other environmental agents which may result in an accumulation of cytogenetic damage with age. Most exposures are chronic and occur at low rates, and conventional cytogenetic methods such as dicentric analysis are not expected to be informative. To understand the extent to which age and lifestyle factors impact the frequency of stable aberrations, we have performed chromosome painting on metaphase-arrested lymphocytes cultured from 47 healthy adults ranging in age from 19 to 77 years, and from umbilical cord blood obtained from eight healthy full-term infants. All subjects had previously been screened to eliminate those who had received significant occupational or accidental exposure to radiation or chemicals, and none had received chemo- or radiotherapy. Due to the infrequent occurrence of stable aberrations in peripheral lymphocytes, we analyzed the equivalent of more than 1100 metaphase cells from each of these 55 people. An average of one cell in 130 (0.77%) was observed to have a translocation or a stable insertion. A significant relationship between stable aberrations and the square of the age is apparent (R2 = 0.69, Y = 0.0615 + 0.000304 age2; p < 0.00001). These results support the hypothesis that stable aberrations accumulate with time, and are likely to integrate adverse environmental exposure.

Adult↗

Plasma levels of 8-methoxypsoralen after topical paint PUVA.

BACKGROUND: Topical PUVA therapy has become a useful alternative for patients who cannot tolerate the systemic side effects of nausea and headache or are concerned about the ophthalmologic risk associated with oral PUVA therapy. However, there is no study to date on the systemic absorption of psoralen after the localized application of topical paint PUVA. OBJECTIVE: This study was designed to assess the plasma level of 8-methoxypsoralen (8-MOP) after paint PUVA therapy for patients with palmoplantar psoriasis or eczema. METHODS: Reverse-phase high-pressure liquid chromatography was used to determine 8-MOP plasma levels in eight patients with palmoplantar psoriasis and two with eczema. Three patients receiving oral PUVA therapy served as the control group. RESULTS: Plasma levels of 8-MOP taken 1, 6, and 24 hours after topical PUVA treatments of patients with palmoplantar psoriasis were undetectable. One patient with hand eczema consistently had detectable 8-MOP levels 1 hour after topical PUVA treatments. CONCLUSION: This report indicates that there is minimal, if any, systemic absorption of 8-MOP after topical PUVA treatment of patients with palmoplantar psoriasis.

Administration, Topical↗

DNA amplifications on chromosomes 7, 9 and 12 in glioblastoma detected by reverse chromosome painting.

Biopsies and cell culture, respectively, of four human glioblastoma multiforme (WHO 4) have been evaluated for gene amplification using reverse chromosome painting. Three of the tumours showed amplified domains within chromosome bands 12q13-15. The exact localisation and extension of the amplified domains, however, varies within this region. Southern blot analysis revealed amplification of the GLI oncogene in two of the glioblastomas which were found to contain amplified domains within 12q13-15. Reverse chromosome painting also identified amplified domains within bands 7q21 and 9p23-24. Amplification within region 9p23-24 has previously not been reported in glioblastoma. The amplified domain encompassing 9p23-24 was detected in the same glioblastoma which contained an amplification unit within bands 12q13-14. These data, together with previous reports, indicate that amplifications are predominantly found on chromosomes 7, 9 and 12 in glioblastoma. In addition, this study provides further evidence that coamplification is not a rare event in glioblastoma.

Chromosomes, Human, Pair 12↗

The relationship between gut contents and supercooling capacity in hatchling painted turtles (Chrysemys picta).

Painted turtles (Chrysemys picta) typically spend their first winter of life in a shallow, subterranean hibernaculum (the natal nest) where they seemingly withstand exposure to ice and cold by resisting freezing and becoming supercooled. However, turtles ingest soil and fragments of eggshell as they are hatching from their eggs, and the ingestate usually contains efficient nucleating agents that cause water to freeze at high subzero temperatures. Consequently, neonatal painted turtles have only a modest ability to undergo supercooling in the period immediately after hatching. We studied the limit for supercooling (SCP) in hatchlings that were acclimating to different thermal regimes and then related SCPs of the turtles to the amount of particulate matter in their gastrointestinal (GI) tract. Turtles that were transferred directly from 26 degrees C (the incubation temperature) to 2 degrees C did not purge soil from their gut, and SCPs for these animals remained near -4 degrees C for the 60 days of the study. Animals that were held at 26 degrees C for the duration of the experiment usually cleared soil from their GI tract within 24 days, but SCPs for these turtles were only slightly lower after 60 days than they were at the outset of the experiment. Hatchlings that were acclimating slowly to 2 degrees C cleared soil from their gut within 24 days and realized a modest reduction in their SCP. However, the limit of supercooling in the slowly acclimating animals continued to decline even after all particulate material had been removed from their GI tract, thereby indicating that factors intrinsic to the nucleating agents themselves also may have been involved in the acclimation of hatchlings to low temperature. The lowest SCPs for turtles that were acclimating slowly to 2 degrees C were similar to SCPs recorded in an earlier study of animals taken from natural nests in late autumn, so the current findings affirm the importance of seasonally declining temperatures in preparing animals in the field to withstand conditions that they will encounter during winter.

Acclimatization↗

Fresco paintings studied by unilateral NMR.

Unilateral NMR has been used to monitor the state of conservation of frescoes in the Vasari's house in Florence. The causes of deterioration of ancient frescoes are varied, which result in the detachment and crumbling of the painted film from the supporting plaster and in the outcropping of salts. Unilateral measurements of Hahn echo performed on such frescoes have allowed a perfect identification of the detachment of the painted film from the plaster. The presence of soluble salts on the pictorial film affects the spin-spin relaxation times, T(2). It is then possible using this technique, to characterize the effect of chemical treatments, of cleansing and consolidation procedures using the distribution of T(2) spin-spin relaxation times.

Journal Article↗

X-ray-sensitivity of lymphocytes of aplastic- and Diamond-Blackfan-anemia patients as detected by conventional cytogenetic and chromosome painting techniques.

Frequencies of spontaneously occurring and X-ray induced, stable and unstable types of chromosome aberrations in peripheral blood lymphocytes from two groups of radiosensitive patients, i.e., aplastic anemia (AA) and Diamond-Blackfan anemia (DBA), were determined. Two types of staining methods, i.e., chromosome painting with two cocktails of chromosome-specific DNA libraries (Nos. 1, 3, X and Nos. 2, 4 and 8), as well as conventional Giemsa staining, were employed. Chromosome painting was done with single and multicolor fluorescence in situ hybridization (FISH). The frequencies of spontaneously occurring chromosome aberrations in AA and DBA patients were not significantly different from healthy individuals. Hypersensitivity to X-rays was seen both in G0 as well as in G2 phase of the cell cycle in lymphocytes from AA and DBA patients, confirming our earlier findings using micronucleus (DBA) and G2 radiosensitivity (AA) assays.

Anemia, Aplastic↗

Painting technique for staining the anterior lens capsule.

We describe a technique of capsule staining called capsule painting. Using a Khokhar capsule painting cannula, the dye is spread directly over the anterior capsule but to no other part of the anterior chamber. There is minimal turbulence in the anterior chamber as there is no need to replace the injected viscoelastic material. The use of minimal dye and viscoelastic material reduces the cost of surgery.

Capsulorhexis↗

Comparative painting of mammalian chromosomes.

Comparative chromosome painting has shown that synteny has been conserved for large segments of the genome in various placental mammals. Advances such as spectral karyotyping and multicolour 'bar coding' lend speed and precision to comparative molecular cytogenetics. Reciprocal chromosome painting and hybridizations with probes such as yeast artificial chromosomes, cosmids, and fibre fluorescence in situ hybridisation allow subchromosomal assignments of chromosome regions and can identify breakpoints of rearranged chromosomes. Advances in molecular cytogenetics can now be used to test the hypothesis that chromosome rearrangement breakpoints in human pathology and in evolution are correlated.

Animals↗

Fatal and non-fatal outcome by accidental intoxication with paint thinner.

Although poisonings (fatal and non-fatal) due to intentional or accidental acute exposure to toluene or toluene mixture fumes have been previously reported in the literature, several issues concerning lethal doses or lasting post-exposure neuropathological impairments still remain unclear. Two male painters (18 and 30 years old) were accidentally exposed to toxic concentrations of paint diluent fumes containing toluene (TL), acetone (ACT) and methyl ethyl ketone (MEK) (60:15:15 w/w/w respectively) during their work in an underground reservoir. Both workers were found unresponsive by colleagues and were immediately transferred to the regional hospital. On admission, the younger man was pronounced dead, while the other remained in the intensive care unit for 3 days and then 4 days in the internal medicine ward. TL, ACT and MEK concentrations in blood samples taken from the survivor on admission were 6.3, 30.6 and 40.5 microg/mL. Postmortem toxicology of the dead worker revealed TL, ACT and MEK blood levels of 12.4, 90.8 and 80.4 microg/mL respectively. The solvent levels in the liver, kidney, lung, brain, testis and gland were also quantified and showed a somewhat similar distribution of the chemicals among these tissues with the highest levels found in the brain and the liver. The fatal and the non-fatal outcome that resulted despite similar intoxication conditions, most probably demonstrates the interindividual tolerance among the painters who also had similar body weights. The surviving painter did not develop any neuropsychological impairment in post-exposure time. The reported case strongly emphasizes the necessity to take precautions when using paint diluents in enclosed spaces.

Journal Article↗