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PubMed2003

Crystallization and preliminary X-ray diffraction analysis of pyranose 2-oxidase from the white-rot fungus Trametes multicolor.

Pyranose 2-oxidase (P2Ox) is a 270 kDa homotetrameric flavoenzyme that catalyzes the oxidation of D-glucose to 2-keto-D-glucose. P2Ox participates in lignin degradation by white-rot fungi and a tentative role of the enzyme is the production of H(2)O(2) for lignin peroxidases. Crystals of Trametes multicolor P2Ox were grown from monomethylether PEG 2000, sodium acetate, MgCl(2) and Ta(6)Br(12). They belong to space group P2(1), with unit-cell parameters a = 99.9, b = 101.7, c = 135.6 A, beta = 90.85 degrees. X-ray diffraction data to 2.0 A resolution were collected using synchrotron radiation. Self-rotation function calculations suggest that the asymmetric unit contains one homotetramer with 222 point-group symmetry.

Basidiomycota↗

PubMedDate not supplied

Detection of cone dysfunction induced by digoxin in dogs by multicolor electroretinography.

It is difficult to detect discrete cone function with the present conventional electroretinography (ERG) examination. In this study, we developed contact electrodes with a built-in color (red (644 nm), green (525 nm), or blue (470 nm)) light source (color LED-electrode), and evaluated an experimental model of digoxin in the dog. First, 17 normal Beagle dogs were used to determine which electrode works well for color ERG measurement on dogs. Then, color ERG was performed on seven normal Beagle dogs at various points during a 14-day period of digoxin administration. A single daily dose of 0.0125 mg/kg/day, which is within the recommended oral maintenance dosage range for dogs, was administered orally for 2 weeks. Ophthalmic examination, measurement of plasma concentration of digoxin, and color ERG examination were performed. On first examination, amplitudes of all responses were significantly (P < 0.01) lower with the red, than with the blue and green electrodes during ERG recording. In ERG using the red electrode, the standard deviation was large. According to these preliminary results, the red electrode was not used in the experimental dog model with digoxin. In the digoxin administrated animals, no significant change was observed in the ophthalmic examination findings. The digoxin level increased steadily throughout the dosing period but was always within the therapeutic range for dogs. In rod ERG, no abnormalities were detected with any electrode. In standard combined ERG, decreased amplitude of the a-wave was detected with every electrode. In single flash cone ERG, prolongation of implicit time was detected by color ERG with the blue and green electrodes. In 30-Hz flicker ERG, decreased amplitude was detected only by color ERG with the blue electrode. The decreased amplitude and prolonged implicit time recovered after termination of digoxin administration. Cone dysfunction induced by digoxin in the dog was revealed by multicolor ERG using blue and green LED-electrodes. Multi-color ERG was useful for detecting cone type-specific dysfunction in the dog.

Administration, Oral↗

PubMed2006

A method for enhancing gunshot residue patterns on dark and multicolored fabrics compared with the modified Griess test.

In using infrared or infrared-enhanced photography to examine gunshot residue (GSR) on dark-colored clothing, the GSR particles are microscopically examined directly on the fabric followed by the modified Griess test (MGT) for nitrites. In conducting the MGT, the GSR is transferred to treated photographic paper for visualization. A positive reaction yields an orange color on specially treated photographic paper. The examiner also evaluates the size of the powder pattern based on the distribution of nitrite reaction sites or density. A false-positive reaction can occur using the MGT due to contaminants or dyes that produce an orange cloud reaction as well. A method for enhancing visualization of the pattern produced by burned and partially unburned powder is by treatment of the fabric with a solution of sodium hypochlorite. In order to evaluate the results of sodium hypochlorite treatment for GSR visualization, the MGT was used as a reference pattern. Enhancing GSR patterns on dark or multicolored clothing was performed by treating the fabric with an application of 5.25% solution of sodium hypochlorite. Bleaching the dyes in the fabric enhances visualization of the GSR pattern by eliminating the background color. Some dyes are not affected by sodium hypochlorite; therefore, bleaching may not enhance the GSR patterns in some fabrics. Sodium hypochlorite provides the investigator with a method for enhancing GSR patterns directly on the fabric. However, this study is not intended to act as a substitute for the MGT or Sodium Rhodizonate test.

Journal Article↗

PubMed2001

Purification and characterization of pyranose oxidase from the white rot fungus Trametes multicolor.

We purified an intracellular pyranose oxidase from mycelial extracts of the white rot fungus Trametes multicolor by using ammonium sulfate fractionation, hydrophobic interaction, ion-exchange chromatography, and gel filtration. The native enzyme has a molecular mass of 270 kDa as determined by equilibrium ultracentrifugation and is composed of four identical 68-kDa subunits as determined by matrix-assisted laser desorption ionization mass spectrometry. Each subunit contains one covalently bound flavin adenine dinucleotide as its prosthetic group. The enzyme oxidizes several aldopyranoses specifically at position C-2, and its preferred electron donor substrates are D-glucose, D-xylose, and L-sorbose. During this oxidation reaction electrons are transferred to oxygen, yielding hydrogen peroxide. In addition, the enzyme catalyzes the two-electron reduction of 1,4-benzoquinone, several substituted benzoquinones, and 2,6-dichloroindophenol, as well as the one-electron reduction of the ABTS [2,2'-azinobis(3-ethylbenzthiazolinesulfonic acid)] cation radical. As judged by the catalytic efficiencies (k(cat)/K(m)), some of these quinone electron acceptors are much better substrates for pyranose oxidase than oxygen. The optimum pH of the pyranose oxidase-catalyzed reaction depends strongly on the electron acceptor employed and varies from 4 to 8. It has been proposed that the main metabolic function of pyranose oxidase is as a constituent of the ligninolytic system of white rot fungi that provides peroxidases with H(2)O(2). An additional function could be reduction of quinones, key intermediates that are formed during mineralization of lignin.

Amino Acid Sequence↗

PubMed1998

Multicolor cytoenzymatic evaluation of dipeptidyl peptidase IV (CD26) function in normal and neoplastic human T-lymphocyte populations.

Dipeptidyl peptidase IV (DPP IV), also identified as the glycoprotein CD26, is a transmembrane 110- to 120-kDa serine aminopeptidase involved in immune responses by influencing T-cell costimulation and by cleaving cytokines. Additionally, CD26 is a nonintegrin receptor that contains a binding site for extracellular matrix and other molecules. In order to further define the expression and functional activity of this membrane exopeptidase in human T cells, we developed a nondisruptive, four-color cytofluorogenic assay that utilizes three separate antibodies to cell-surface molecules (e.g., CD4/CD8/CD26 and CD19/CD56/CD26) along with a rhodamine 110-conjugated dipeptide substrate that allows the measurement of DPP IV activity in phenotypically defined cells. We found normal human thymi to have notable differences in time-dependent DPP IV activity among the thymocyte subsets defined by their CD4/CD8 phenotype, with CD4-/CD8- thymocytes containing less DPP IV activity than cells expressing CD4 and/or CD8 (i.e., maturing). CD26 positivity was moderately intense in thymocytes and tended to identify cells with higher DPP IV activity. The four-color technique was also used to examine mature peripheral blood lymphocytes, along with an assortment of leukemias and transformed T-cell lines. These experiments revealed that while DPP IV was consistently evident in normal T cells, neoplastic T cells could vary in their expression patterns. Furthermore, the presence (or intensity) of surface CD26 in some abnormal T cells and certain normal peripheral blood mononuclear cells was separable from the level of DPP IV measured intracellularly. Our results established that multicolor cytofluorographic analysis can be a practical means to measure DPP IV activity in various human cell populations. Furthermore, we found that DPP IV activity could vary in T cells according to their differentiation status and that under certain circumstances surface CD26 expression can be disassociated from the level of measured enzyme (i.e., DPP IV) activity.

Cells, Cultured↗

PubMed1999

Aneuploidy in pig sperm: multicolor fluorescence in situ hybridization using probes for chromosomes 1, 10, and Y.

The objective of this research was to develop chromosome-specific probes for use in evaluating aneuploidy in boar spermatozoa through the application of fluorescence in situ hybridization (FISH) technology. A multicolor FISH method was developed to detect aneuploidy in the sperm of boars using DNA probes specific for small regions of chromosomes 1, 10, and Y. The average frequencies of sperm with disomy for chromosomes 1, 10, and Y were 0.075%, 0.067%, and 0.094%, respectively. The incidence of disomy did not differ significantly by chromosome. The average frequencies of diploidy were 0.177% for 1-1-10-10 and 0.022% for Y-Y-10-10. Thus, the incidence of overall diploidy (1-1-10-10) was significantly higher than that of disomy for the chromosomes examined (P < 0.01 for disomy of the autosomes and P < 0.05 for disomy of the Y chromosome). No significant age or breed effects on disomy and diploidy rates and no significant interindividual variations in disomy or diploidy were found. The observed level of numerical chromosome aberrations in pig sperm appear to be within the range of the baseline frequencies reported so far in men.

Aneuploidy↗

PubMed2000

Spontaneous frequencies of aneuploid and diploid sperm in 10 normal Chinese men: assessed by multicolor fluorescence in situ hybridization.

Many studies have been published establishing the background frequencies of disomic and diploid sperm in normal men by fluorescence in situ hybridization (FISH) analysis, with highly significant variance among the reports. Besides interdonor heterogeneity and differences in the experimental protocols used, the question of inherent differences in chromosome malsegregation and meiotic arrest among different geographic and ethnic groups of donors has been raised. In this study, multicolor FISH analysis was carried out on semen samples from 10 nonsmoking, nondrinking Chinese men from the People's Republic of China. The results were compared to FISH data on 10 nonsmoking, nondrinking Canadians under the same experimental conditions, in the same laboratory. A total of 200,497 sperm was scored in the Chinese donors and compared to 202,320 sperm from Canadian donors. Approximately 10,000 sperm per chromosome probe per donor were analyzed. The mean hybridization efficiency was 99.99%. The frequencies of X-bearing and Y-bearing sperm were not significantly different from the expected 50% for each individual and for the combined data from all donors (49.73% vs. 49.46%, P = 0.3946). The mean disomy frequencies (range) were 0.07% (0.02%-0.12%) for chromosome 13, 0.18% (0.09%-0.19%) for chromosome 21, 0.05% (0. 01%-0.09%) for 24,XX, 0.02% (0.01%-0.06%) for 24,YY, and 0.29% (0. 13%-0.49%) for 24,XY. The mean diploidy frequency (range) was 0.38% (0.22%-0.73%) for 13-21 hybridizations and 0.32% (0.07%-0.70%) for XY hybridizations. Highly significant interdonor heterogeneity was found for diploidy (P = 0.0000) and for XY disomy (P = 0.0011), but no age effect was observed in any category of disomic or diploid sperm. The data reported here show no marked differences in disomy and diploidy frequencies between the mainland Chinese and Canadian groups, if donor heterogeneity is taken into account.

Adolescent↗

PubMed2001

Reconstruction of the female Gorilla gorilla karyotype using 25-color FISH and multicolor banding (MCB).

The origin of the human and great ape chromosomes has been studied by comparative chromosome banding analysis and, more recently, by fluorescence in situ hybridization (FISH), using human whole-chromosome painting probes. It is not always possible, however, to determine the exact breakpoints and distribution or orientation of specific DNA regions using these techniques. To overcome this problem, the recently developed multicolor banding (MCB) probe set for all human chromosomes was applied in the present study to reanalyze the chromosomes of Gorilla gorilla (GGO). While the results agree with those of most previous banding and FISH studies, the breakpoints for the pericentric inversion on GGO 3 were defined more precisely. Moreover, no paracentric inversion was found on GGO 14, and no pericentric inversions could be demonstrated on GGO 16 or 17.

Animals↗

PubMed2002

Multicolor chromosome banding (MCB) with YAC/BAC-based probes and region-specific microdissection DNA libraries.

Multicolor chromosome banding (MCB) allows the delineation of chromosomal regions with a resolution of a few megabasepairs, i.e., slightly below the size of most visible chromosome bands. Based on the hybridization of overlapping region-specific probe libraries, chromosomal subregions are hybridized with probes that fluoresce in distinct wavelength intervals, so they can be assigned predefined pseudo-colors during the digital imaging and visualization process. The present study demonstrates how MCB patterns can be produced by region-specific microdissection derived (mcd) libraries as well as collections of yeast or bacterial artificial chromosomes (YACs and BACs, respectively). We compared the efficiency of an mcd library based approach with the hybridization of collections of locus-specific probes (LSP) for fluorescent banding of three rather differently sized human chromosomes, i.e., chromosomes 2, 13, and 22. The LSP sets were comprised of 107 probes specific for chromosome 2, 82 probes for chromosome 13, and 31 probes for chromosome 22. The results demonstrated a more homogeneous coverage of chromosomes and thus, more desirable banding patterns using the microdissection library-based MCB. This may be related to the observation that chromosomes are difficult to cover completely with YAC and/or BAC clones as single-color fluorescence in situ hybridization (FISH) experiments showed. Mcd libraries, on the other hand, provide high complexity probes that work well as region-specific paints, but do not readily allow positioning of breakpoints on genetic or physical maps as required for the positional cloning of genes. Thus, combinations of mcd libraries and locus-specific large insert DNA probes appear to be the most efficient tools for high-resolution cytogenetic analyses.

Chromosome Banding↗

PubMed2003

CABAND: Classification of aberrations in multicolor banded chromosomes.

A system is presented to describe aberrations in chromosomes painted with the mBAND methodology. The CABAND (classification of aberrations in multicolor banded chromosomes) system is based on the fact that only banded parts of aberrations and not DAPI stained parts can be described accurately.

Chromosome Aberrations↗

PubMed2003

Multicolor spectral karyotyping of rat chromosomes.

Rat and mouse have become important animal models to study various human diseases such as cancer. Cytogenetic analysis of the respective karyotypes is frequently required to investigate the causative genetic defects and especially neoplastic cells often show complex chromosome aberrations and many different marker chromosomes. However, structural homogeneity of the chromosomes in these species as well as less pronounced differences in banding patterns make it difficult to assign genetic abnormalities to certain chromosomes by conventional banding techniques. Here we report for the first time the successful application of multicolor spectral karyotyping (SKY) to rat chromosomes, which allows unequivocal identification of all rat chromosomes with the exception of chromosomes 13 and 14 in different colors, thus enabling the elucidation of even complex rearrangements in the rat karyotype. Flow-sorted chromosome specific painting probes for all 22 rat chromosomes (20 autosomes, X, and Y) were combinatorially labeled by a set of five different fluorochromes and hybridized in situ to metaphase spreads of a healthy rat, to diakineses from testicular material, and to cells from a rat FAO hepatoma cell line. Measuring the complete spectrum at each image point by using the SpectraCube((R)) spectral imaging system and respective computer software allowed identification of the individual rat chromosomes by their specific emission spectra. Classification algorithms in the analysis software can then display the rat chromosomes in specific pseudo-colors and automatically order them in a karyotype table. After its successful application to human and mouse chromosomes, spectral karyotyping of rat chromosomes now also allows cytogenetic screening of the complete rat genome by a single hybridization.

Animals↗

PubMed2004

mBAND: a high resolution multicolor banding technique for the detection of complex intrachromosomal aberrations.

Precise breakpoint definition of chromosomal rearrangements using conventional banding techniques often fails, especially when more than two breakpoints are involved. The classic banding procedure results in a pattern of alternating light and dark bands. Hence, in banded chromosomes a specific chromosomal band is rather identified by the surrounding banding pattern than by its own specific morphology. In chromosomal rearrangements the original pattern is altered and therefore the unequivocal determination of breakpoints is not obvious. The multicolor banding technique (mBAND, see Chudoba et al., 1999) is able to identify breakpoints unambiguously, even in highly complex chromosomal aberrations. The mBAND technique is presented and illustrated in a case of intrachromosomal rearrangement with seven breakpoints all having occurred on one chromosome 16, emphasizing the unique analyzing power of mBAND as compared to conventional banding techniques.

Chromosome Aberrations↗

PubMed2004

The hierarchically organized splitting of chromosome bands into sub-bands analyzed by multicolor banding (MCB).

To clarify the nature of chromosome sub-bands in more detail, the multicolor banding (MCB) probe-set for chromosome 5 was hybridized to normal metaphase spreads of GTG band levels at approximately 850, approximately 550, approximately 400 and approximately 300. It could be observed that as the chromosomes became shorter, more of the initial 39 MCB pseudo-colors disappeared, ending with 18 MCB pseudo-colored bands at the approximately 300-band level. The hierarchically organized splitting of bands into sub-bands was analyzed by comparing the disappearance or appearance of pseudo-color bands of the four different band levels. The regions to split first are telomere-near, centromere-near and in 5q23-->q31, followed by 5p15, 5p14, and all GTG dark bands in 5q apart from 5q12 and 5q32 and finalized by sub-band building in 5p15.2, 5q21.2-->q21.3, 5q23.1 and 5q34. The direction of band splitting towards the centromere or the telomere could be assigned to each band separately. Pseudo-colors assigned to GTG-light bands were resistant to band splitting. These observations are in concordance with the recently proposed concept of chromosome region-specific protein swelling.

Chromosome Banding↗

PubMed2005

Three cases with rare interstitial rearrangements of chromosome 1 characterized by multicolor banding.

In this report, we describe three unrelated patients with similar symptoms such as mental retardation, growth delay and multiple phenotypic abnormalities. GTG-banding analysis revealed karyotypes with add(1p) in two cases and an add(1q) in the third. Fluorescence in situ hybridization (FISH) analysis using high resolution multicolor banding (MCB) characterized the aberrations of the abnormal chromosomes 1 as a (sub)terminal duplication and inverted duplications, respectively. Although three different chromosomal regions i.e. 1p36.1, 1p36.2-->1p31.3 and 1q41-->1q44 were involved, all three patients had similar patterns of dysmorphic findings. These cases demonstrate the power of MCB in the characterization of small interstitial chromosomal aberrations and resulted in the characterization of three previously unreported congenital chromosome 1 rearrangements.

Abnormalities, Multiple↗

PubMed2006

Combined multicolor-FISH and immunostaining.

The combination of multicolor-FISH and immunostaining produces a powerful visual method to analyze in situ DNA-protein interactions and dynamics. Representing one of the major technical improvements of FISH technology, this method has been used extensively in the field of chromosome and genome research, as well as in clinical studies, and serves as an important tool to bridge molecular analysis and cytological description. In this short review, the development and significance of this method will be briefly summarized using a limited number of examples to illustrate the large body of literature. In addition to descriptions of technical considerations, future applications and perspectives have also been discussed focusing specifically on the areas of genome organization, gene expression and medical research. We anticipate that this versatile method will play an important role in the study of the structure and function of the dynamic genome and for the development of potential applications for medical research.

Animals↗

PubMed2006

Multicolor fluorescence in situ hybridization (FISH) applied to FISH-banding.

During the last decade not only multicolor fluorescence in situ hybridization (FISH) using whole chromosome paints as probes, but also numerous chromosome banding techniques based on FISH have been developed for the human and for the murine genome. This review focuses on such FISH-banding techniques, which were recently defined as 'any kind of FISH technique, which provide the possibility to characterize simultaneously several chromosomal subregions smaller than a chromosome arm. FISH-banding methods fitting that definition may have quite different characteristics, but share the ability to produce a DNA-specific chromosomal banding'. While the standard chromosome banding techniques like GTG lead to a protein-related black and white banding pattern, FISH-banding techniques are DNA-specific, more colorful and, thus, more informative. For some, even high-resolution FISH-banding techniques the development is complete and they can be used for whole genome hybridizations in one step. Other FISH-banding methods are only available for selected chromosomes and/or are still under development. FISH-banding methods have successfully been applied in research in evolution- and radiation-biology, as well as in studies on the nuclear architecture. Moreover, their suitability for diagnostic purposes has been proven in prenatal, postnatal and tumor cytogenetics, indicating that they are an important tool with the potential to partly replace the conventional banding techniques in the future.

Bone Marrow Cells↗

PubMed2006

Multicolor chromosome bar codes.

Chromosome bar codes are multicolor banding patterns produced by fluorescence in situ hybridization (FISH) with differentially labeled and pooled sub-regional DNA probes. These molecular cytogenetic tools facilitate chromosome identification and the delineation of both inter- and intra-chromosomal rearrangements. We present an overview of the various conceptual approaches which can be largely divided into two classes: Simple bar codes designed for chromosome identification and complex bar codes for high resolution aberration screening of entire karyotypes. We address the issue of color redundancy and how to overcome this limitation by complementation of bar codes with whole chromosome painting probes.

Chromosome Aberrations↗

PubMed2006

Multicolor-FISH applied to resolve complex chromosomal changes in a case of T-ALL (FAB L2).

We report on a patient with a clinically diagnosed acute lymphoblastic leukemia (ALL) with partial unrecorded complex translocation events especially involving chromosomes 5, 9 and 18. At the GTG-band level the karyotype was abnormal in 20% of the analyzed cells. The complex karyotype was studied in more detail by spectral karyotyping (SKY) and multicolor banding (MCB) to characterize it in more detail. Thus, the karyotype could be described very accurately and in summary three different clones were detected, reflecting a high rate of karyotypic evolution in this patient.

Adolescent↗