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PubMed1994

Multicolor flow cytometric analysis of the CD45 antigen provides improved lymphoid cell discrimination in bone marrow and tissue biopsies.

Samples from bone marrow or non-hematopoietic tissue such as solid organ biopsies often contain an excess of non-leukocytes exhibiting lymphocyte-like light scatter characteristics, making it sometimes difficult to define satisfactory light scatter lymphocyte gates. To circumvent this, we describe here a multiparametric method of identifying lymphoid cells by expression of the CD45 antigen, in conjunction with light scatter parameters. A 'third color'-conjugated anti-CD45 antibody was included with every FITC/PE double staining, thereby permitting live or list mode analysis gating on CD45 positive cells. The triple-staining technique was applied to (a) human bone marrow, showing that special attention has to be given to the enumeration of B cells, and (b) to liver biopsies, where gating on CD45 fluorescence and orthogonal light scatter was shown to clearly resolve all lymphocyte subsets from debris. All cell types examined in tissue biopsies as well as T and NK cells in bone marrow were best distinguished by gating on bright CD45 expression in conjunction with low orthogonal light scatter, while accurate identification of marrow B cells relied upon including all levels of CD45 intensity. The multicolor gating procedure, aimed mainly at immune-monitoring of non-malignant tissues, is applicable to most kinds of single cell samples, and may prove to be an aid for lymphocyte gating in cases where leukocyte populations are not clearly resolved on a light scatter basis alone.

Antibodies, Monoclonal↗
PubMed1986

Satiation restores brood care motivation in the female mouthbrooder Pseudocrenilabrus multicolor (Cichlidae).

The maternal mouthbrooder Pseudocrenilabrus multicolor takes up her eggs during spawning and carries them continuously in her mouth for 10-11 days at 27 degrees C. Then the young-by that time able to swim-are released from the mouth. The duration of mouthbrooding is controlled by an internal timing program in the mother [12]. In non-first-spawners brood care motivation (= readiness to take up brood into the mouth and keep it there) disappears within a few hours after brood removal. At the same time hunger-which is reduced to half of the normal level when brood care motivation is present-increases to the normal level [15]. In females without brood care motivation, satiation by food intake or by a water solution of 15 mg/l diethylpropion (an anorectic amphetamine-derivative), restores brood care motivation during a sensitive period of approximately 11 days after spawning. The point of release of adopted brood from the mouth depends on the duration of previous brood deprivation, but not on the state of brood care motivation nor on the hunger state during brood deprivation. The results suggest that above a certain level hunger inhibits brood care motivation. By contrast, the hunger state seems not to influence internal timing of mouthbrooding.

Animals↗
PubMed1992

Regional assignment of the human keratin 5 (KRT5) gene to chromosome 12q near D12S14 by PCR analysis of somatic cell hybrids and multicolor in situ hybridization.

Keratin 5 is the major type II keratin of the basal cells of epidermis and of other stratified epithelia. With its type I partner, keratin 14, it constitutes a major fraction of the cytoskeleton of the basal cells. Because the inheritance of epidermolysis bullosa simplex, a disease of epidermal basal cell fragility, was mapped in one family to chromosome 12q close to D12S14, we undertook to localize the gene for keratin 5. Polymerase chain reaction analysis of somatic cell hybrids mapped the keratin 5 gene to chromosome 12, and multicolor fluorescence in situ hybridization localized it to 12q very near D12S14. This sublocalization exemplifies the utility of in situ physical localization in assessing the candidacy of genes thought to underlie inherited disorders.

Animals↗
PubMed2004

Combined spectral karyotyping, multicolor banding, and microarray comparative genomic hybridization analysis provides a detailed characterization of complex structural chromosomal rearrangements associated with gene amplification in the osteosarcoma cell line MG-63.

The advancement of fluorescence in situ hybridization-based assays has permitted more refined delineation of chromosomal loci involved in complex chromosomal rearrangements (CCRs) and gene amplification. In this detailed molecular cytogenetic analysis, spectral karyotyping (SKY), multicolor banding (mBAND) analysis, and microarray comparative genomic hybridization (CGH) were used to refine the analysis of chromosomes with amplifications and small intrachromosomal rearrangements such as inverted duplications and interstitial deletions present in the osteosarcoma cell line MG-63. SKY analysis has limited resolving power to delineate cryptic chromosomal rearrangements, so mBAND assays were performed for a subset of chromosomes (i.e., 6, 8, 17, and 20). Of the 10 clonal CCRs analyzed in detail with mBAND, 5 were found to have rearrangements between 8q24 and either 6p23 approximately pter or 6p21, with multiple copies of this translocation inserted at various sites in the different chromosomes. In two CCRs, 6p21 and 8q24 generated an alternating pattern of mBAND probe hybridization, indicating the presence of a large coamplified repeat unit within homogeneously staining regions. Microarray CGH analysis demonstrated focal high-level amplification of 8q23 approximately q24, 6p22 approximately pter, and 6p21, in agreement with the pattern of chromosome subband gains identified with mBAND. Thus, sequential SKY, mBAND, and microarray CGH provided a comprehensive description of some of the intricate chromosomal aberrations present in the complex MG-63 karyotype and permitted reconstruction of the fine structure of the genomic rearrangements, thus providing some important mechanistic clues concerning the details of the amplification process in tumors.

Bone Neoplasms↗
PubMed2005

Multicolor fluorescence in situ hybridization characterization of cytogenetically polyclonal hematologic malignancies.

Several different investigations and methodologies have provided data supporting a monoclonal origin of neoplasia. For example, the vast majority of neoplastic disorders are cytogenetically monoclonal. Occasionally, however, clones with unrelated karyotypic anomalies are found, as, for example, in approximately 2% of acute myeloid leukemias (AML), myelodysplastic syndromes (MDS), and chronic myeloproliferative disorders (CMD). Whether such a cytogenetic polyclonality represents a polyclonal origin or whether different clones share a submicroscopic primary change, indicating a monoclonal origin, remains to be elucidated. Our objective was to ascertain if cryptic aberrations can be found in cytogenetically polyclonal hematologic malignancies using multicolor fluorescence in situ hybridization (M-FISH). Fourteen AML, MDS, and CMD cases were investigated. In none of these was a cryptic aberration found, common to all subclones, although the karyotypes were revised in two AMLs and one MDS. Thus, all malignancies were still classified as polyclonal after the M-FISH analyses. Based on the present results, we conclude that M-FISH, in general, does not reveal primary cryptic aberrations supporting a monoclonal origin of cytogenetically polyclonal hematologic malignancies.

Adult↗
PubMed2006

Identification of chromosome aberrations in esophageal cancer cell line KYSE180 by multicolor fluorescence in situ hybridization.

Analysis of chromosomal changes in esophageal squamous cell carcinoma (ESCC) can illuminate the molecular mechanisms underlying the development and progression of this cancer, which is among the 10 most common malignant tumors. Cell lines are better suited than surgical samples for chromosome analysis in this cancer. This study used multicolor fluorescence in situ hybridization (M-FISH) to characterize the molecular cytogenetics of ESCC in cell line KYSE180. Two pools of 12-color whole-chromosome painting probes were designed, and two rounds of FISH were performed on the same metaphase spreads. Loss of DNA copy number was observed at 4p, 5q, 6q, 9, 10p, 12p, 13, 14p, 15p, 18p, 18q, 20, 22, and Y. Chromosomal gains and translocations occurred at the entire or part of 1, 2p, 3, 4p, 5p, 5q, 6p, 7, 8, 10q, 11, 12q, 14q, 16, 17q, 19, and Xp. Seven derivative chromosomes (5, 8, 12, 14, 14, 14, and 17) presented complex translocations, each involving three or four chromosomes. No chromosomes 9, 13, or Y were detected. These results add significant information to the existing karyotype description of KYSE180 and provide detailed cytogenetic background data for appropriate use of the cell line.

Carcinoma, Squamous Cell↗
PubMed2005

Enumeration and characterization of virus-specific B cells by multicolor flow cytometry.

To better characterize B cell responses induced to influenza virus, we developed an assay to directly quantify and characterize virus-specific B cells. We used purified and biotinylated whole virus as well as the major influenza virus surface antigen, hemagglutinin (HA) to label virus-specific B cells induced by immunization of mice with whole influenza virus in adjuvant. Immunization with adjuvant alone caused non-specific binding of whole virus to a large number of B cells in the draining lymph nodes as assessed by flow cytometry. This precluded the use of whole virus as a specific staining reagent. In contrast, staining with bromelain-cleaved purified and biotinylated influenza virus HA identified a small population of B cells (roughly 1%) only in the draining lymph nodes of virus-immunized mice. FACS-purification and subsequent ELISPOT analysis showed that HA-labeled B cells contained the vast majority of virus-specific antibody-secreting cells at day 10 after immunization. Overall, virus-specific antibody-secreting cells comprised roughly 10% of the HA-labeled cells. Using HA-staining in conjunction with 8-color flow cytometry we further demonstrated that close to 90% of the HA-labeled cells were CD19+ IgD- CD23- CD24high CD38low germinal center B cells, many of which had incorporated bromodeoxyuridine, indicating recent cell division in vivo. We conclude that viral HA can be used in conjunction with cell surface and intracytoplasmic stains in multicolor flow cytometry to provide detailed phenotypic and functional information on virus HA-specific B cells.

Animals↗
PubMed2006

Multicolor fluorescence imaging for early detection of the hypersensitive reaction to tobacco mosaic virus.

The physiological status of plants can nowadays be promptly monitored with non-invasive methods. This opens the possibility to continuously follow-up plant performance and permits to detect stress-induced deviations presymptomatically. Upon stress, plants may synthesize specific compounds, depending on the causal agent. Such compounds may alter the absorption of the light impinging on plant leaves, hence the spectrum of reflected, re-emitted, and transmitted light changes. UV-excited fluorescence imaging specifically allows visualization of the accumulation of phenolic compounds, e.g. those associated with the hypersensitive response to pathogens. By using imaging at regular intervals (time-lapse series) of tobacco mosaic virus (TMV) infection in resistant tobacco we aimed at the description and quantification of the kinetics of blue-green fluorescence compared to the visual development of the disease. Presymptomatic responses to TMV infection were observed with a multicolor fluorescence and reflectance imaging setup. The onset of increases in blue-green and chlorophyll fluorescence were comparable in timing, although further symptom development was strikingly different. Compounds known to accumulate during the hypersensitive response and displaying blue-green fluorescence revealed different dynamics of fluorescence evolution in time. The multichannel imaging system permitted to discern the key components salicylic acid and scopoletin. In contrast, for the compatible interaction between TMV and non-resistant tobacco, no presymptomatic responses were detected on inoculated leaves. This work proves the potential of multispectral imaging to unveil stress-associated signatures, and the power of blue-green fluorescence imaging to monitor accumulation of secondary compounds.

Fluorescence↗
PubMed2005

Simultaneous enumeration of viable Enterobacteriaceae and Pseudomonas spp. within three hours by multicolor fluorescence in situ hybridization with vital staining.

A new means of rapidly and simultaneously counting viable phylogenetically different bacteria was developed. The cyanine dimer dye, BOBO-3 that selectively stains bacteria with damaged membranes were used to evaluate bacterial viability based on membrane integrity. Viable Enterobacteriaceae and Pseudomonas spp. could be selectively detected within three hours using multicolor fluorescence in situ hybridization (FISH) following BOBO-3 staining (BOBO3-FISH).

Bacteriological Techniques↗
PubMed2003

Multicolor deconvolution microscopy of thick biological specimens.

One limitation in understanding disease at the cellular level has been the inability to efficiently analyze DNA on a cell-to-cell basis within the natural tissue context. However, DNA analyses at a single-cell resolution should be instrumental for the understanding of cancer cell biology, cancer evolution, for chromosomal mosaic analysis and rare cell events, and should provide otherwise inaccessible information on essential biological processes. Here we present a fluorescence in situ hybridization-based multicolor deconvolution technique for three-dimensional microscopy. We use up to seven different color channels for probe detection, which allows the simultaneous high-resolution localization of multiple point-like sources within a biological specimen with a thickness of up to 30 micro m. In addition, a DNA counterstain is used for volume labeling of the nuclei offering the opportunity for a simultaneous segmentation of nuclei. Furthermore, as the instrumentation consists of a standard fluorescence microscope it represents a low-cost method as compared to confocal microscopy.

Centromere↗
PubMed2003

Identification of the covalent flavin adenine dinucleotide-binding region in pyranose 2-oxidase from Trametes multicolor.

We present the first report on characterization of the covalent flavinylation site in flavoprotein pyranose 2-oxidase. Pyranose 2-oxidase from the basidiomycete fungus Trametes multicolor, catalyzing C-2/C-3 oxidation of several monosaccharides, shows typical absorption maxima of flavoproteins at 456, 345, and 275 nm. No release of flavin was observed after protein denaturation, indicating covalent attachment of the cofactor. The flavopeptide fragment resulting from tryptic/chymotryptic digestion of the purified enzyme was isolated by anion-exchange and reversed-phase high-performance liquid chromatography. The flavin type, attachment site, and mode of its linkage were determined by mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy of the intact flavopeptide, without its prior enzymatic degradation to the central aminoacyl moiety. Mass spectrometry identified the attached flavin as flavin adenine dinucleotide (FAD). Post-source decay analysis revealed that the flavin is covalently bound to histidine residue in the peptide STHW, consistent with the results of N-terminal amino acid sequencing by Edman degradation. The type of the aminoacyl flavin covalent link was determined by NMR spectroscopy, resulting in the structure 8alpha-(N(3)-histidyl)-FAD.

Basidiomycota↗
PubMed2001

Multicolored protein conformation states in the photocycle of transducer-free sensory rhodopsin-I.

Sensory rhodopsin-I (SRI), a phototaxis receptor of archaebacteria, is a retinal-binding protein that exists in the cell membrane intimately associated with a signal-transducing protein (HtrI) homologous to eubacterial chemotaxis receptors. Transducer-free sensory rhodopsin-I (fSRI), from cells devoid of HtrI, undergoes a photochemical cycle kinetically different from that of native SRI. We report here on the measurement and analysis of the photochemical kinetics of fSRI reactions in the 350-750-nm spectral range and in a 10(-7) s to 1 s time window. The lack of specific intermolecular interactions between SRI and HtrI results in early return of the ground form via distinct branching reactions in fSRI, not evident in the photocycle of native SRI. The chromophore transitions are loosely coupled to protein structural transitions. The coexistence of multiple spectral forms within kinetic intermediates is interpreted within the concept of multicolored protein conformational states.

Archaeal Proteins↗
PubMed2002

Multicolor imaging of Ca(2+) and protein kinase C signals using novel epifluorescence microscopy.

Dynamic changes in intracellular free Ca(2+) concentrations ([Ca(2+)](i)s) control many important cellular events, including binding of Ca(2+)-calmodulin (Ca(2+)-CaM) and phosphorylation by protein kinase C (PKC). The two signals compete for the same domains in certain substrates, such as myristoylated alanine-rich PKC-substrate (MARCKS). To observe the convergence and relative time of arrival of CaM and PKC signals at their shared domain of MARCKS, we need to image cells that are loaded with more than two fluorescent dyes at a reasonable speed. We have developed a simple and powerful multicolor imaging system using conventional fluorescence microscopy. The epifluorescence configuration uses a glass reflector and rotating filter wheels for excitation and emission paths. As it is free of dichroic (multichroic) mirrors, multiple fluorescence images can be acquired rapidly regardless of the colors of fluorophores. We visualized Ca(2+)-CaM and PKC together with the dynamics of their common target, MARCKS, in single live cells. Receptor-activation resulted in translocation of MARCKS from the plasma membrane to cytosol through its phosphorylation by PKC. By observing fluorescence resonance energy transfer, we also obtained direct evidence that Ca(2+)-CaM binds MARCKS to drag it away from the membrane in circumstances when Ca(2+)-mobilization predominates over PKC activation.

Calcium↗
PubMed2000

Enzymatic synthesis of fucose-containing disaccharides employing the partially purified alpha-L-fucosidase from Penicillium multicolor.

The alpha-L-Fucp-(1 --> 3)-D-GlcpNAc disaccharide structure is a vital core unit of the oligosaccharide components of glycoconjugates isolated from human milk and blood group substances. Alpha-L-Fucosidase from Penicillium multicolor catalyses the transfer of L-fucose from donor structures such as alpha-L-FucpOpNP and alpha-L-FucpF to various GlcpNAc derivatives and Glcp, forming alpha-(1 --> 3) linkages. The synthesis of several biologically relevant disaccharides including alpha-L-Fucp-(1 --> 3)-alpha-D-GlcpNAcOMe, alpha-L-Fucp-(1 --> 3)-alpha-D-GlcpNAcOAll, alpha-L-Fucp-(1 --> 3)-beta-D-GlcpNAcOAll, alpha-L-Fucp-(1 --> 3)-D-GlcpNAc and alpha-L-Fucp-(1 --> 3)-D-Glcp has been achieved in up to 34% yields by application of this enzyme.

Animals↗
PubMed1998

An alpha-L-fucosidase from Penicillium multicolor as a candidate enzyme for the synthesis of alpha (1-->3)-linked fucosyl oligosaccharides by transglycosylation.

A new alpha-L-fucosidase was partially purified from the culture broth of Penicillium multicolor, which was available commercially as a freeze dried powder by the name of Lactase-P. This enzyme catalysed the transglycosylation of fucose residue of p-nitrophenyl-alpha-L-fucopyranoside to give alpha-L-Fuc-(1-->3)-D-Glc or alpha-L-Fuc-(1-->3)-D-GlcNAc regioselectively. This enzyme was more stable in the organic co-solvents than the alpha-fucosidase from Aspergillus niger, which was also proposed previously by us as an enzyme to produce fucosyl oligosaccharides.

Chromatography, Gel↗
PubMed1998

Prenatal detection of aneuploidy by directly labeled multicolored probes and interphase fluorescence in situ hybridization.

OBJECTIVE: To detect aneuploidy of chromosomes 13, 18, 21, X, and Y with use of new, directly labeled, multicolored, commercially available DNA probes from interphase cells of amniotic fluid (AF). MATERIAL AND METHODS: The hybridization sites of the five probes were validated by metaphase analysis. The fluorescence in situ hybridization (FISH) normal range was determined from a series of normal AF specimens and tested on a series of normal and abnormal specimens. RESULTS: The hybridization efficiencies of the five probes were 100%. The mean AF interphase disomic signal patterns for chromosomes 13, 18, 21, XX, and XY were 95.9%, 89.1%, 94.3%, 94.7%, and 98.7%, respectively. Of a total of 508 cases analyzed, 211 were aneuploid. All cases were correctly identified and no false results occurred (in comparison with karyotypic analysis), although maternal cell contamination was relatively common. CONCLUSION: Clinical screening for aneuploidy of chromosomes 13, 18, 21, X, and Y from interphase AF cells is possible with use of these probes and FISH. Cases of maternal cell contamination and mosaicism necessitate cautious interpretation. The FISH procedure is recommended for screening of common aneuploidies, followed by a complete chromosome analysis to detect anomalies.

Amniotic Fluid↗
PubMed2000

Spectral karyotyping and multicolor fluorescence in situ hybridization reveal new tumor-specific chromosomal aberrations.

Spectral karyotyping (SKY), multiple fluorescence in situ hybridization (M-FISH), cross-species color banding (Rx-FISH), multicolor chromosome banding, and other labeling techniques and strategies have been recent comprehensive technical developments in the field of molecular cytogenetics. The immediate goals of these methods are (1) to reliably characterize complex chromosomal rearrangements present in tumor karyotypes; (2) to screen for new tumor-specific chromosomal aberrations; (3) to improve genetic classification systems of different tumor types in correlation with clinical data, treatment regimens, detection of minimal residual disease, and prognosis; and (4) to identify new target regions for gene identification strategies. We present a brief overview of the different methods, including summaries of numerous published and submitted papers detailing specific cytogenetic aberrations associated with leukemias and lymphomas. To date, 640 tumor cases have been analyzed by SKY, including 410 hematologic malignancies, 146 solid tumors, and 45 mouse tumors.

Animals↗
PubMed2002

Multicolor-FISH analysis of a natural killer cell line (NK-92).

Relatively little is known about the cytogenetics of natural killer (NK) neoplasms, and the emergence of recurrent structural alterations involving specific chromosomal breakpoints is still in its infancy. This gap has doubtless hampered identification of the oncogene alterations posited to underly NK tumors. We describe in detail the cytogenetic rearrangements present in a cytotoxic NK cell line (NK-92) established from a patient with large granular lymphocyte (LGL) lymphoma. The NK-92 cell line is one of very few cytotoxic NK cell lines described and the first to be used clinically. Cytogenetic analysis was performed independently using two multicolor-fluorescence in situ hybridization (M-FISH) systems, the first M-FISH study of a cell line derived from a NK neoplasm. Several non-random cytogenetic features previously reported in NK cells were, thus, identified, including rearrangements of chromosomes 7 and 17, along with breakpoints at 11q23, 12q12 and 8p22/23. FISH revealed that NK-92 cells carry multiple rearrangements with distinct breakpoints at 8p resembling those previously described in NK lymphoma. Our data strengthen the claim of NK-92 to model NK neoplasms and highlight this cell line as a potential resource for mining relevant oncogenic changes therein.

Chromosome Banding↗