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PubMed2001

Application of multicolor fluorescent in situ hybridization for enhanced characterization of chromosomal abnormalities in congenital disorders.

OBJECTIVE: To determine the efficacy of multicolor fluorescent in situ hybridization (M-FISH), which paints each chromosome in a unique color, for identification of congenital derivative and marker chromosomes. MATERIAL, METHODS AND CASES: Commercially available M-FISH probes were used to label each chromosome in a specific fluorescent color. Six representative cases involving derivative chromosomes, markers, and subtle anomalies were analyzed by M-FISH. RESULTS: Three familial, rather subtle derivative chromosomes were identified by M-FISH with relative ease. A small ring that was unidentifiable by banded-chromosome analysis was identified by M-FISH. A case of a subtle telomeric anomaly could not be resolved without the use of telomeric-specific probes. The M-FISH results were confirmed by individual chromosome-specific painting probes. CONCLUSION: M-FISH was helpful for identifying a wide range of congenital chromosomal anomalies. However, for subtle chromosomal abnormalities, use of locus-specific probes may be necessary.

Adult↗
PubMedDate not supplied

Multicolor fluorescence technique to detect apoptotic cells in advanced coronary atherosclerotic plaques.

Apoptosis occurring in atherosclerotic lesions has been suggested to be involved in the evolution and the structural stability of the plaques. It is still a matter of debate whether apoptosis mainly involves vascular smooth muscle cells (vSMCs) in the fibrous tissue or inflammatory (namely foam) cells, thus preferentially affecting the cell-poor lipid core of the atherosclerotic plaques. The aim of the present investigation was to detect the presence of apoptotic cells and to estimate their percentage in a series of atherosclerotic plaques obtained either by autopsy or during surgical atherectomy. Apoptotic cells were identified on paraffin-embedded sections on the basis of cell nuclear morphology after DNA staining and/or by cytochemical reactions (TUNEL assay, immunodetection of the proteolytic poly (ADP-ribose) polymerase-1 [PARP-1] fragment); biochemical procedures (identifying DNA fragmentation or PARP-1 proteolysis) were also used. Indirect immunofluorescence techniques were performed to label specific antigens for either vSMCs or macrophages (i.e., the cells which are most likely prone to apoptosis in atherosclerotic lesions): the proper selection of fluorochrome labeling allowed the simultaneous detection of the cell phenotype and the apoptotic characteristics, by multicolor fluorescence techniques. Apoptotic cells proved to be less than 5% of the whole cell population, in atherosclerotic plaque sections: this is, in fact, a too low cell fraction to be detected by widely used biochemical methods, such as agarose gel electrophoresis of low-molecular-weight DNA or Western-blot analysis of PARP-1 degradation. Most apoptotic cells were of macrophage origin, and clustered in the tunica media, near or within the lipid-rich core; only a few TUNEL-positive cells were labeled for antigens specific for vSMCs. These results confirm that, among the cell populations in atherosclerotic plaques, macrophage foam-cells are preferentially involved in apoptosis. Their death may decrease the cell number in the lipid core and generate a possibly defective apoptotic clearance: the resulting release of matrix-degrading enzymes could contribute to weakening the fibrous cap and promote the plaque rupture with the risk of acute ischemic events, while increasing the thrombogenic pultaceous pool of the plaque core.

Apoptosis↗
PubMed1995

Isochromophilones III-VI, inhibitors of acyl-CoA:cholesterol acyltransferase produced by Penicillium multicolor FO-3216.

New azaphilones named isochromophilones III-VI were isolated from the culture broth of Penicillium multicolor FO-3216 as inhibitors of acyl-CoA: cholesterol acyltransferase (ACAT). Their structures were elucidated by NMR and other spectroscopic analyses. The IC50 values of isochromophilones III, IV, V and VI for ACAT activity in an enzyme assay using rat liver microsomes were calculated to be 110, 50, 50 and 120 microM, respectively.

Animals↗
PubMed1995

Isochromophilones I and II, novel inhibitors against gp120-CD4 binding produced by Penicillium multicolor FO-2338. I. Screening, taxonomy, fermentation, isolation and biological activity.

Isochromophilones I and II, the first novel gp120-CD4 binding inhibitors of microbial origin, were isolated from a cultured broth of a soil fungus designated as Penicillium multicolor FO-2338. These compounds were obtained as yellow powders from the cultured broth together with the known related compounds sclerotiorin, ochrephilone and rubrorotiorin. Isochromophilones I and II (C23H25O5Cl and C22H27O4Cl, respectively) have an azaphilone skeleton and a chlorine atom. Isochromophilones strongly inhibited gp120-CD4 binding (IC50: 6.6 and 3.9 microM, respectively), but the other related compounds did not. Isochromophilone II inhibited significantly HIV replication in peripheral human lymphocytes at 25 microM.

Antiviral Agents↗
PubMed1995

Isochromophilones I and II, novel inhibitors against gp120-CD4 binding produced by Penicillium multicolor FO-2338. II. Structure elucidation.

The structures of isochromophilones I and II, new gp120-CD4 binding inhibitors isolated from a cultured broth of Penicillium multicolor FO-2338, were elucidated by NMR experiments. Both of compounds have an azaphilone skeleton substituted by a chlorine atom at C-5 and a side chain, 3,5-dimethyl-1,3-heptadien at C-3. Additionally, isochromophilone I has a gamma-lactone ring, and isochromophilone II has 2-oxopropyl moiety instead of a gamma-lactone ring.

Antiviral Agents↗
PubMed1999

Fluoro-Gold: An alternative viability stain for multicolor flow cytometric analysis.

BACKGROUND: The viability stains propidium iodide (PI) and 7-amino-actinomycin D (7-AAD) are excited at 488 nm, as are the commonly used antibody conjugates fluorescein isothiocyanate (FITC), phycoerythrin (PE), and cyanine 5 dye covalently coupled to R-phycoerythrin (RPE-Cy5). When excited by a single laser, spectral overlap in the emission of PI and 7-AAD with RPE-Cy5 precludes the use of these viability stains for three-color immunophenotyping, particularly when evaluating low levels of marker expression in viable target cells. The ultraviolet excitable dye hydroxystilbamidine methanesulfonate (Fluoro-Gold, or FG) binds to DNA at the A-T-rich regions of the minor groove in permeabilized or dead cells. We assessed the suitability of this dye as a viability stain. METHODS: The ability of FG to detect nonviable cells in fresh and cryopreserved human apheresed peripheral blood cells was compared with that of PI and 7-AAD. The stability of FG staining and the effects of dye and cell concentration on the discrimination of nonviable cells was determined by measuring changes in the median fluorescence of viable and nonviable cells. RESULTS: FG labeling at dye concentrations of 2-8 microM is stable for at least 3 h over a wide range of cell concentrations (4 x 10(5) to 4 x 10(7) cells/ml). Costaining studies and linear regression analysis show that cell viability as determined by FG is strongly correlated with estimates using PI (r = 0.9636) and 7-AAD (r = 0.9879). CONCLUSIONS: FG is a reliable, alternative viability stain that can be used in conjunction with fluorochromes including FITC, PE, and RPE-Cy5 for multicolor analysis using dual-laser instruments.

Antigens, CD↗
PubMed2001

Characterization of chromosome aberrations in salivary gland tumors by FISH, including multicolor COBRA-FISH.

Fluorescence in situ hybridization (FISH), including COBRA-FISH, was used to characterize 11 salivary gland tumors that had been investigated by banding analysis. Five cases were pleomorphic adenoma (PA), three were adenoid cystic carcinoma, and one case each was mucoepidermoid carcinoma, carcinoma ex-pleomorphic adenoma (CaPA), and adenocarcinoma. All 11 cases were selected on the basis that they had shown rearrangement of 6q or 9p or had unresolved aberrations after karyotyping. The COBRA-FISH and FISH analyses led to a revised karyotype in all informative cases and made it possible to clarify almost all chromosomal rearrangements occurring in the tumors. Of particular note were the confirmation of the existence of 6q deletions, a common change in salivary gland carcinomas, and the demonstration that a seemingly balanced t(6;9) resulted in del(6q). Other rearrangements that were revealed by FISH included amplification of 12q sequences (MDM2 and CDK4) in one PA. We also investigated the status of the PLAG1 gene in four cases (one PA, one CaPA, one adenoid cystic carcinoma, and one mucoepidermoid carcinoma) with 8q12 rearrangements. Only in the former two cases were the FISH results compatible with intragenic rearrangements. Overall, the results of the study show that, even with good banding quality and in karyotypes of modest complexity, much new information will be gained by supplementing the banding analysis with a multicolor FISH approach, such as COBRA-FISH.

Adult↗
PubMed2002

[From "monocolor" karyotype to "multicolor" karyotype: applications of M-Fish in hematology and oncology].

Since the establishment of human karyotype in 1956, human cytogenetic has quickly progressed. The description of the Philadelphia chromosome in 1960 led up to new applications of cytogenetic in the fields of hematology and oncology. The initial techniques allowed only uniform staining of chromosomes, limiting the detection of most structural rearrangements. Many approaches aimed to gain a better knowledge of chromosomal structure, a better understanding of rearrangements, and a better identification of the chromosomes were developed: autoradiography, banding techniques, electronic microscopy. Since 1980, new developments in clinical cytogenetic and molecular biology have occurred. In situ labeling using non-radioactive probes onto chromosomes and nuclei was developed: fluorescence in situ hybridization (Fish) was born. Fish allows detecting many chromosomal abnormalities of number and/or structure. The major limitation of this technique is that its use should be based on known indications for the choice of the probe. Multicolor karyotype (M-Fish or Sky), the most recent development of Fish on metaphase spreads, allows to overcome this limit. As shown here in three examples, M-Fish allows to describe precisely complex rearrangements in hematological malignancies and solid tumors. Finally, if no metaphase is available, comparative genomic hybridization (CGH) can be performed to detect and simultaneously localize on chromosomes gains or losses in genomic DNA.

Carcinoma, Transitional Cell↗
PubMed2002

Microdissection based high resolution multicolor banding for all 24 human chromosomes.

The multicolor-banding (MCB) approach allows the differentiation of chromosome region specific areas at the band and sub-band level and is based on region-specific microdissection libraries producing changing fluorescence intensity ratios along the chromosomes. The latter are used to assign different pseudocolors to specific chromosomal regions. We present the complete set of 138 region-specific microdissection libraries for the entire human genome and the resulting MCB patterns for all human chromosomes at the 450-550 band level. In the present work, the creation and handling of the microdissection libraries is detailed for the first time. Additionally, the unique possibilities of the MCB technique to adjust the pseudocolor bands according to the necessities of the studied case is presented in exemplarity. In conclusion, the MCB-technique is a high resolution alternative to other FISH based chromosome banding approaches and suited to clarify, which changes appeared in complex chromosomal rearrangements.

Chromosome Banding↗
PubMed2002

Evidence for interphase DNA decondensation transverse to the chromosome axis: a multicolor banding analysis.

In the present study a multicolor banding (MCB) analysis was performed to address the up to now unrequited question in which direction with respect to the axis chromosomes decondense in interphase. It could be demonstrated, that i) MCB produces a similar banding pattern in interphase as in metaphase; ii) that no complete decondensation and dispersion appears, which is in concordance with the concept of chromosome territories; and iii) chromosome decondensation happens square to chromosome axis. The presented data are important for a better understanding of nuclear architecture, however, further studies are required.

Chromosome Banding↗
PubMed2002

A complex translocation event between the two homologues of chromosomes 5 leading to a del(5)(q21q33) as a sole aberration in a case clinically diagnosed as CML: characterization of the aberration by multicolor banding.

We report on a patient with a clinically diagnosed Philadelphia negative chronic myelogenous leukemia (CML) with a so far unrecorded complex translocation event between the two homologue chromosomes 5. At the GTG-band level the karyotype was normal, apart from an enlarged chromosome 5 and an extremely shortened second chromosome 5. Both derivative chromosomes 5 consisted exclusively of #5 derived material as proven by 24-color FISH. To characterize the complex aberration in more detail the multicolor banding (MCB) technique using a chromosome 5 specific probe set was applied. Using this DNA-based high resolution banding procedure, the karyotype could be described as 46,XX,del(5)(pterright curved arrow q12::q33right curved arrow qter),ins(5)(pterright curved arrow q15::q12right curved arrow q21::q21right curved arrow qter). In consequence, the aberration leads to a partial deletion of the long arm of chromosome 5: del(5)(q21q33), which would not have been identified using conventional banding techniques or 24-color FISH.

Aged↗
PubMed2002

The effect of multicolored machinable ceramics on the esthetics of all-ceramic crowns.

STATEMENT OF PROBLEMS: Computer-aided design/computer-assisted machining systems offer the possibility of fabricating restorations from one machinable ceramic block. Whether multishaded blocks improve esthetic results and are a viable alternative to individually stained ceramics has not been fully determined. PURPOSE: The aim of this investigation was to examine the effect of multishaded blocks on the esthetic appearance of all-ceramic CEREC crowns and compare these crowns with single-shade and stained restorations. MATERIAL AND METHODS: Ten subjects were included in this study. For each subject, 6 different crowns were milled with the use of a CEREC machine. One crown was milled from each of the following machinable ceramic materials: CEREC Vitablocs Mark II in classic colors; Vitablocs Mark II in 3D-Master colors; Vitablocs Mark II in either classic or 3D-Master colors, with additional staining; Megadenta Bloxx multishaded; Mark II experimental multilayer; and an experimental multilayer leucite ceramic. Three independent examiners assessed the esthetic appearance of crowns fabricated to match each subject's anterior tooth shade. A scale of 1 to 6 was used to score the shade match and esthetic adaptation of each crown, with 1 representing excellent characteristics and 3.5 serving as the threshold for clinical acceptability. The examiners' scores were averaged, and the mean values were analyzed with the Wilcoxon signed rank test (P<or=.05). RESULTS: Only individually stained Mark II restorations achieved a mean score below the threshold of 3.5 for all 10 subjects. These restorations were significantly more esthetic (P<or=.05) than restorations fabricated from all other materials except the Mark II experimental multilayer ceramic. The second best results were obtained for crowns made from single-shaded Mark II 3D-Master blocks: 6 out of 10 restorations were scored below 3.5. Two of the layered materials (Mark II experimental and Bloxx) followed with 5 acceptable restorations out of 10. CONCLUSION: Within the limitations of this study, the results provide no evidence that multicolored machinable ceramics improve the esthetics of all-ceramic crowns.

Aluminum Silicates↗
PubMedDate not supplied

Chromosomal aberrations of multiple myeloma in Chinese patients at diagnosis: a study by combined G-banding and multicolor spectral karyotyping.

Cytogenetic investigation of multiple myeloma (MM) has been difficult by conventional methods and most of the data have been derived from western population where incidence of MM is much higher as compared to that of Asians. The current study represents the first report of chromosomal aberrations of multiple myeloma in Chinese. We investigated 25 consecutive Chinese patients with MM for chromosomal aberrations at diagnosis using G-banding and multicolor spectral karyotyping (SKY). Of the 21 patients successfully analyzed by G-banding, 11 patients revealed cytogenetic abnormalities showing complex numerical and structural aberrations, which were further characterized with SKY. An abnormal karyotype significantly associated with blastic MM was observed. Consistent with the western literature, structural rearrangements involving chromosomes 1, 6, 8, 19, numerical abnormalities of gains in chromosomes 9, 3, and 5, and losses in chromosomes 13 and 14 were observed. However, there were notably higher incidences of -22/22q- (4/11) and structural aberrations of chromosome X but a lower incidence of -X. The biological implications of these findings, if confirmed, deserve further evaluation.

Adult↗
PubMed2003

Detailed Hylobates lar karyotype defined by 25-color FISH and multicolor banding.

A comprehensive and detailed comparative chromosome map of the white-handed gibbon (Hylobates lar = HLA) has been established by hybridizing the recently developed complete human multicolor banding (MCB) probe set on metaphase chromosomes of a male HLA lymphoblastoid cell line. Thus, it was possible to precisely determine the breakpoints and distribution plus orientation of specific DNA-regions in this cytogenetically highly rearranged species compared to Homo sapiens (HSA). In general, the obtained results are in concordance with previous molecular-cytogenetic studies. In this study all 71 breakpoints present in HLA compared to HSA could be determined exactly. This study is a valuable complement to our knowledge on the phylogeny of huminoid chromosomes.

Animals↗
PubMed2003

[Sublethal effects of insecticides on fecundity of multicolored Asian ladybird Harmonia axyridis].

The effects of sublethal doses of 6 insecticides (imidacloprid, rotenone, fenvalerate, abamectin, pirimicarb and azadirachtin) on the fecundity of multicolored Asian ladybird Harmonia axyridis were determined respectively. The results showed that in all treatments, the daily average number of eggs per female ladybird was about 40-55. Ladybirds pretreated with imidacloprid by contact and feeding methods, and with rotenone by feeding method produced significantly fewer eggs, compared with control. But the accumulative oviposition of ladybirds pretreated with 6 insecticides at sublethal level was no significant differences, compared with untreated ladybirds. The hatching rates of the eggs laid by ladybirds pretreated with 6 insecticides by two methods were all lower than that of control. The accumulative survivals from egg to pupal stage of all treatments decreased significantly. The developmental duration from egg to pupa of those treated with imidacloprid by two methods and with fenvalerate by contact method prolonged significantly, while that of those treated with rotenone by two methods and with fenvalerate, abamectin, pirimicarb, and azadirachtin by feeding method shortened obviously, but that of those treated with rotenone and abamectin by contact method had no differences with control. After the eggs were immersed with sublethal concentrations of imidacloprid, rotenone, fenvalerate and abamectin, the successful hatching rates were all lower than that of control, and the development periods prolonged clearly. The 1st instar larval survival rates of those treated with fenvalerate decreased significantly, and the survivals from 2nd instar larval to pupal stage also reduced to some extent. The total developmental duration of those treated with rotenone and fenvalerate prolonged significantly. These findings suggested that the effects of insecticides at low doses on the fecundity of ladybird were very complicated. The class, action mode, toxicity and exposure route of insecticides could affect the results of experiments.

Animals↗
PubMed2004

[Study on the mechanism of oocyte aneuploidy formation by multicolor fluorescence in situ hybridization].

OBJECTIVE: To investigate the mechanism of oocyte aneuploidy formation. METHODS: The unfertilized oocytes were fixed 1-3 days after oocyte retrieval. Multicolor fluorescence in situ hybridization (M-FISH) was performed according to the Vysis protocol to check the chromosome status in oocytes by using centromeric enumerator probes for chromosomes 16, 18 and locus-specific probes for chromosomes 13, 21 and 22. RESULTS: 47% oocytes were found to be normal, while 53 % oocytes were abnormal. Nondisjunction was found in 22(18%) oocytes, unbalanced predivision of chromatids in 15(12%) oocytes, balanced predivision of chromatids in 45(36%) oocytes. The balanced predivision rate in oocytes aged in vitro>24h was much higher than that in oocytes aged in vitro<or=24h (P<0.01). CONCLUSION: Both nondisjunction, balanced and unbalanced predivision of chromatids are involved in the oocyte aneuploidy formation. Balanced predivision is related to the time in culture.

Adult↗
PubMed2006

Molecular cytogenetic characterization of the mouse cell line WMP2 by spectral karyotyping and multicolor banding applying murine probes.

The Moloney murine leukemia virus-transformed suspension cell line WMP2 is derived from wild mice (Mus musculus) of the WMP/WMP strain. These mice carry nine pairs of metacentric Robertsonian translocation chromosomes. As the chromosomes of the wild-type mouse are all acrocentric, metaphase spreads of the WMP2 cells seam to be highly suited for physical gene mapping. Here we studied the WMP2 line using spectral karyotyping (SKY) combined with new established mouse specific multicolor banding (mcb) probes for the chromosomes X, 3, 4, 6 and 18. SKY revealed that the WMP2 cell line developed further four derivative chromosomes. After application of mcb five previously unrecognizable intrachromosomal rearrangements with 9 breakpoints were detected for the studied chromosomes.

Animals↗
PubMed2005

[Diagnosis of numerical chromosomal aberrations in the cells of spontaneous abortions by multicolor fluorescence in situ hybridization (MFISH)].

According to different estimates, as high as 15-20% of all the pregnancies result in spontaneous abortions (SA) at different gestational periods. Identification of abnormalities leading to SA is of great importance for practical medicine, mainly for medical genetic counseling of married couples with impaired reproductive function. The diagnosis of chromosomal aberrations on the basis of SA materials is known to have a number of methodological difficulties. The present paper deals with the identification of numerical anomalies in the SA material by multicolor fluorescence in situ hybridization (MFISH). This technique using an original collection of DNA probes for chromosomes 1, 9, 13/21, 14/22, 15, 16, 18, X, and Y was applied to the study of chromosomal aberrations in 224 spontaneous abortion specimens. Numerical chromosomal aberrations were found in 122 (54.5%) cases. The cells of all the studied specimens exhibited aneuploidy of chromosome X in 17% cases; chromosome 16 in 12%, chromosomes 13/21 in 5.8%, chromosomes 14/22 in 4.9%, chromosomes 9 and 18 in 1.3% (each), chromosome 15 in 0.9%, chromosome 1 in 0.45%. Polyploidy was detected in 13.3% of cases; concomitant abnormalities were found in 7 cases. Analysis of the findings has led to the conclusion that MFISH can be successfully used in the diagnosis of numerical chromosomal aberrations of CA cells.

Abortion, Spontaneous↗