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PubMed1977

Changes in the distribution of a major fibroblast protein, fibronectin, during mitosis and interphase.

The distribution of a major fibroblast protein, fibronectin, was studied by immunofluorescence and immunoscanning electron microscopy in cultures of human and chicken fibroblasts during different phases of the cell cycle. The main findings were: (a) In interphase cells, the intensity of surface-associated fibronectin fluorescence correlated with that of intracellular fibronectin fluorescence. (b) The intensity of the fluorescence of both surface-associated and intracellular fibronectins was not changed in cells that were synthesizing DNA. (c) Mitotic cells had reduced amounts of surface-associated but not of intracellular fibronectin. The surface fibronectin that remained on meta-, ana-, or telophase cells had a distinct punctate distribution and was also localized to strands attaching the cells to the substratum. Fibronectin strands first reappeared on the surface of flattening cytoplasmic parts of telophase cells. (d) Fibronectin was also detected in extracellular fibrillar material on the growth substratum, particularly around dividing cells. Thus, surface-associated fibrillar fibronectin was present during G(1), S, and G(2) but in cells undergoing mitosis the distribution was altered and the amount appeared to be reduced. The observations on the distribution of surface-associated fibronectin suggest that rather than being involved in growth control this fibronectin plays a structural role in interactions of cells with the environment.

Animals↗
PubMed1978

Intranuclear actin bundles induced by dimethyl sulfoxide in interphase nucleus of Dictyostelium.

Electron microscopic evidence demonstrated that dimethyl sulfoxide (DMSO) induces formation of giant intranuclear microfilament bundles in the interphase nucleus of a cellular slime mold, Dictyostelium. These giant bundles are approximately giant bundles are approximately 3 micrometer long, 0.85 micrometer wide, and composed of microfilaments 6 nm in diameter. Studies in which glycerinated cells were used showed that these microfilaments bind rabbit skeletal muscle heavy meromyosin, forming typical decorated "arrowhead" structures, and that this binding can be reverted by Mg-adenosine triphosphate. These data verify that the intranuclear microfilaments are the contractile protein actin, and that DMSO affects intranuclear actin, inducing the formation of such giant bundles. The intranuclear actin bundles appear at any developmental stage in two different species of cellular slime molds after treatment with DMSO. The native form of the intranuclear actin molecules and their possible functions are discussed, and it is proposed that the contractile protein has essential functions in the cell nucleus.

Actins↗
PubMed1984

Widespread cellular distribution of MAP-1A (microtubule-associated protein 1A) in the mitotic spindle and on interphase microtubules.

In the accompanying paper (Bloom, G.S., T.A. Schoenfeld, and R.B. Vallee, 1983, J. Cell Biol. 98:320-330), we reported that microtubule-associated protein 1 (MAP 1) from brain comprises multiple protein species, and that the principal component, MAP 1A, can be detected in both neuronal and glial cells by immunofluorescence microscopy using a monoclonal antibody. In the present study, we sought to determine the cellular and subcellular distribution of MAP 1A in commonly used cultured cell systems. For this purpose we used immunofluorescence microscopy and immunoblot analysis with anti-MAP 1A to examine 18 types of mammalian cell cultures. MAP 1A was detected in every culture system examined. Included among these were cells of mouse, rat, Chinese hamster, Syrian hamster, Potoroo (marsupial), and human origin derived from a broad variety of tissues and organs. Anti-MAP 1A consistently labeled mitotic spindles and stained cytoplasmic fibers during interphase in most of the cultures. These fibers were identified as microtubules by co-localization with tubulin in double-labeling experiments, by their disappearance in response to colchicine or vinblastine, and by their reorganization in response to taxol. The anti-MAP 1A stained microtubules in a punctate manner, raising the possibility that MAP 1A is located along microtubules at discrete foci that might represent sites of interaction between microtubules and other organelles. Verification that MAP 1A was, indeed, the reactive material in immunofluorescence microscopy was obtained from immunoblots. Anti-MAP 1A stained a band at the position of MAP 1A in all cultures examined. These results establish that MAP 1A, a major MAP from brain, is widely distributed among cultured mammalian cells both within and outside of the nervous system.

Alkaloids↗
PubMed2002

Measuring surface-area-to-volume ratios in soft porous materials using laser-polarized xenon interphase exchange nuclear magnetic resonance.

We demonstrate a minimally invasive nuclear magnetic resonance (NMR) technique that enables determination of the surface-area-to-volume ratio (S/V) of soft porous materials from measurements of the diffusive exchange of laser-polarized 129Xe between gas in the pore space and 129Xe dissolved in the solid phase. We apply this NMR technique to porous polymer samples and find approximate agreement with destructive stereological measurements of S/V obtained with optical confocal microscopy. Potential applications of laser-polarized xenon interphase exchange NMR include measurements of in vivo lung function in humans and characterization of gas chromatography columns.

Lasers↗
PubMed2000

Rapid confirmation of previously detected prenatal mosaicism by fluorescence in situ hybridization in interphase uncultured amniocytes.

Fluorescence in situ hybridization (FISH) of chromosome-specific probes to interphase uncultured amniocytes was performed in cases in which follow-up amniocenteses were done for confirmation of previously detected mosaicism. FISH results were informative in all seven cases included in the study, and confirmed by subsequent cytogenetic analysis. FISH analysis provides rapid results for referral physicians and in most cases reassurance for patients within 24 hours of the follow-up aminocentesis. Although FISH studies are not considered accurate in determining a primary diagnosis of mosaicism in uncultured cells, the analysis is accurate and clinically useful when the diagnosis is known and mosaicism involving a specific chromosome needs to be confirmed in follow-up testing.

Amniocentesis↗
PubMed1997

Interphase cytogenetic (in situ hybridization) analysis of astrocytomas using archival, formalin-fixed, paraffin-embedded tissue and nonfluorescent light microscopy.

Astrocytomas contain nonrandom chromosomal abnormalities that recently have been correlated with shortened patient survival. Two frequently reported aberrations are trisomy 7 and monosomy 10. We assessed the numerical complement of chromosomes 7 and 10 in formalin-fixed, paraffin-embedded brain biopsy tissue from 28 diffuse astrocytomas by in situ hybridization using a nonfluorescent enzymatic detection system. Clinical follow-up of at least 5 years was available in 26 cases (93%). Monosomy 10 was identified in 7 cases (25%): astrocytoma, 1 case; anaplastic astrocytoma, 1 case; and glioblastoma, 5 cases. Trisomy 7 was identified in 11 cases (39%): astrocytoma, 5 cases; glioblastoma, 6 cases. Multivariate analysis revealed that monosomy 10 was the most statistically significant negative predictor of patient survival. Numerical chromosomal abnormalities are detectable in astrocytomas in archival tissue using interphase cytogenetics and nonfluorescent light microscopy. Although larger studies are required, our data suggest that potentially useful prognostic information may be obtained with this approach.

Adult↗
PubMed1998

Comparative analysis of interphase FISH and RT-PCR to detect bcr-abl translocation in chronic myelogenous leukemia and related disorders.

The t(9;22)(q34;q1 1) between the abl and bcr genes plays a pivotal role in the diagnosis and pathogenesis of chronic myelogenous leukemia (CML). Its detection is routinely accomplished by Southern blot analysis and karyotyping. Interphase fluorescence in situ hybridization (FISH) and reverse transcriptase-polymerase chain reaction (RT-PCR) are emerging molecular techniques that offer viable alternatives. We analyzed 40 samples of peripheral blood and bone marrow (CML, 16; acute myelogenous leukemia, 6; acute lymphoblastic leukemia [ALL], 1; chronic lymphoblastic leukemia, 2; myelodysplasias, 4; myeloproliferative disorders, unclassified, 3; nonleukemic hematologic malignancies, 3; hypercellular bone marrow, 1; normal control samples, 2; and K562 cell line samples, 2) for the presence of bcr-abl fusion gene and its messenger RNA (mRNA) transcript by FISH and RT-PCR, respectively. We compared the results with results of Southern blot analysis and karyotyping when available. Cost analysis was performed. Thirty-three samples were evaluable by FISH; 14 of 14 evaluable CML samples and one ALL sample were positive for bcr-abl by FISH (100%). The other 15 evaluable samples were negative; 16 of 16 (100%) and 13 of 16 (81%) of CML cases were positive for bcr-abl mRNA by RT-PCR (chemiluminescent blot method) and RT-PCR (colorimetric method), respectively. The ALL sample was positive by both RT-PCR methods. All other samples were negative by RT-PCR (chemiluminescent blot method), and all but 1 case of myeloproliferative disorder tested negative by RT-PCR (colorimetric method). We conclude the utility of FISH and RT-PCR is associated with certain limitations, such as insufficient RNA for RT-PCR and the occasional absence of internal positive FISH control signals. However, each procedure offers (with a high concordance rate) a specific and cost-effective alternative to Southern blot analysis and karyotyping and improved turnaround time for the detection of bcr-abl fusion gene or its mRNA transcript.

Blotting, Southern↗
PubMedDate not supplied

Prenatal diagnosis from fetal urine in bladder outlet obstruction: success rates for traditional cytogenetic evaluation and interphase fluorescence in situ hybridization.

PURPOSE: To determine the optimal approach to the prenatal chromosome analysis of fetal urine from fetuses with bladder outlet obstruction. METHODS: Retrospective evaluation of traditional cytogenetic and interphase fluorescence in situ hybridization (FISH) analysis on fetal urine specimens from fetuses with bladder outlet obstruction. RESULTS: Traditional cytogenetic analysis was successful on 71 (95%) of 75 samples, and FISH was informative on 20 (65%) of 31 specimens. The combination of traditional cytogenetic analysis and FISH yielded a 96% diagnostic success rate. The mean turnaround time was 8 days (range 5-14) for traditional cytogenetic analysis and 1.6 days (range 1.0-4.0) for FISH. Chromosome abnormalities were detected in 6 (7.9%) of 76 pregnancies. CONCLUSION: Traditional cytogenetic analysis achieves a high success rate (95%) and is superior to FISH for chromosome evaluation of fetal urine. However, FISH, when informative, can complement traditional cytogenetics as it will expeditiously rule out common trisomies in fetuses with bladder outlet obstruction.

Female↗
PubMed1994

Loss of Microtubules in the Interphase Cells of Onion (Allium cepa L.) Root Tips from the Cell Cortex and Their Appearance in the Cytoplasm after Treatment with Cycloheximide.

As part of a project to investigate the mechanism of cortical microtubule (MT) alignment, we examined the effects of cycloheximide (CHM) on cortical MTs in the root tip cells of Allium cepa L. Results show that although a preprophase band of MTs remained in the cell cortex, interphase MTs disappeared from the cortical cytoplasm and then appeared concomitantly in the inner cytoplasm when the rate of de novo protein synthesis was reduced with CHM (11-360 [mu]M for 2 h)

Journal Article↗
PubMed1992

In situ detection of supernumerary aberrations of chromosome-specific repetitive DNA targets in interphase nuclei in human melanoma cell lines and tissue sections.

The use of non-radioactive in situ hybridization (ISH) with chromosome-specific repetitive DNA probes to study genomic changes, aneuploidy, and heterogeneity during melanocytic tumor progression, relies on its applicability to non-mitotic interphase nuclei, present in cell suspensions and tissue sections. Therefore, we studied the feasibility of detecting numerical aberrations with respect to the (peri-) centromere regions of chromosomes 1 and 7 in intact nuclei of two human melanoma cell lines with different metastatic behavior in nude mice. In addition, we used paraffin sections from xenograft lesions, obtained by inoculation of these cell lines in nude mice (subcutaneous tumors and spontaneous lung metastases). Paraffin sections from the original primary cutaneous melanoma (with a subepidermal and a dermal part) and two loco-regional metastases were also studied, one of which was the source for the cell lines. These cells and tissues represent examples of materials used in different approaches to the study of melanocytic tumor progression. Regarding the targeted sequences, ISH analysis showed that both cell lines were heterogeneous and aneuploid. The results correlated well with those obtained by ISH on metaphase spreads. Differences between the lines, which could not be detected by flow-cytometric or conventional karyotyping analysis, included data suggestive of a polyploid subpopulation and an extra copy of chromosome 7 in the metastasizing cell line. The polyploid population could be detected also in the paraffin sections of the corresponding subcutaneous xenografts and lung metastases in the mice. Both areas in the patients' primary melanoma could be evaluated separately and showed similar supernumerary aberrations of the chromosome-specific targets. These abnormalities matched those found in both metastases. Our results demonstrate that ISH can be used to visualize genomic abnormalities at the single-cell level in melanocytic nuclei in their natural context, which makes it a promising tool in the histopathology of melanocytic lesions and in the study of melanocytic tumor progression.

Aged↗
PubMed2006

Quantitative misorientation characteristics of interphase boundaries in composites.

Specific crystallographic correlations between neighbouring grains in composites were established by the use of selected area electron diffraction in the transmission electron microscope. However, it was the development of orientation mapping techniques that made it possible to obtain a quantitative description of the distribution of boundaries between the grains of both the same phase and different phases. This study shows that orientation topography measurements made by electron backscatter diffraction in the scanning electron microscope allowed determination of the crystallographic relationships between grains of different phases. An alumina-based composite with a content of 10 vol% tungsten carbide was chosen for investigations. Misorientation distribution functions were calculated to describe the distribution density of misorientations with respect to the nearest neighbouring measured point located in the grain of the second phase. The analysis of misorientation distribution functions permitted the evaluation of preferences for some special crystallographic correlations between the grains of composite matrix and inclusions as well as shares of interphase boundaries characterized by those correlations.

Journal Article↗
PubMed1976

Biochemical and electron-microscopic evidence that the subunit structure of Chinese-hamster-ovary interphase chromatin is conserved in mitotic chromosomes.

Biochemical and electron microscopic studies demonstrate that the subunit structure of Chinese hamster ovary cell interphase chromatin is conserved in mititic chromosomes. Digestion of purified chromosomes or nuclei with micrococcal nuclease produces DNA in discrete size classes, as visualized by polyacrylamide gel electrophoresis, which are common to the two materials. Early in digestion the DNA fragments are integral multiples of a monomer approximately 177 base pairs in length, whereas after extensive digestion the remaining DNA fragments migrate ahead of the monomer position. The size of the repeating DNA unit was confirmed as being smaller than that produced by micrococcal nuclease digestion of rat liver nuclei by direct comparison. Electron microscopy of partially unravelled chromosomes at low ionic strength shows tightly packed spheres (nucleosomes) approximately 12 nm in diameter which are often arranged as linear chains. Chromosomal material prepared for electron microscopy after varying extents of micrococcal nuclease digestion is composed of fragments containing pregressively fewer nucleosomes, which parallels the loss of high DNA multimer bands in gel electrophoresis. Material unravelled from chromosomes in the presence of NaCl consists of nucleosomes packed packed in a different configuration which suggests the origin of higher order structures in chromosomes.

Cell Line↗
PubMed1995

Interphase cytogenetics of gastric carcinoma: fluorescence in situ hybridization (FISH) applied to cells obtained from formalin-fixed paraffin-embedded tissues.

The interphase cytogenetics in formalin-fixed and paraffin-embedded gastric cancer tissues were examined by fluorescence in situ hybridization (FISH) with alpha-satellite DNA probes. Two gastric carcinoma cell lines, TMK-1 and MKN-28, were first analyzed cytogenetically. Of 25 TMK-1 cell karyotypes, chromosome 7 showed trisomy and chromosome 17 showed disomy in 18 cells. Most MKN-28 cells showed disomy of both chromosomes 7 and 17. Suspensions of singly isolated TMK-1 and MKN-7 cells were obtained from the cultured cells, and from paraffin-embedded tissue specimens fixed with formalin for 0, 1, 3 and 5 days obtained from xenotransplanted tumors in nude mice. The numbers of chromosomes 7 and 17 analyzed with the karyotypic preparations coincided well with those determined by FISH, even in the paraffin-embedded specimens. The number of tumor cells showing no signals, however, increased in the specimens after 5 days formalin fixation. In 10 surgically removed gastric carcinomas, the predominant signal number for chromosomes 7 and 17 in the cells of paraffin-embedded tissues was two (disomy), except in one papillary carcinoma, which was trisomic for chromosome 7. Large subpopulations (more than 20%) showing trisomy were found in four cases for chromosome 7 and in five cases for chromosome 17. A higher frequency of trisomy was found in well differentiated than in poorly differentiated carcinomas. These findings suggest that the FISH technique is a useful tool for detecting chromosomal aberrations in gastric adenocarcinoma cells, even in paraffin-embedded specimens, as long as the tissues are fixed with formalin for an appropriate time.

Adenocarcinoma↗
PubMed1971

Y-fluorescence of interphase nuclei, especially circulating lymphocytes.

Application of a fluorescence technique for the detection of the human Y chromosome in interphase nuclei indicates that the best results may be obtained from the study of lymphocytes in blood smears. The most satisfactory Y-fluorescence is obtained with the use of buffered quinacrine mustard dihydrochloride. The method can be complemented by other standard techniques to obtain a clear idea of the sex-chromosome complement of the individual. The application of this technique to clinical and chromosomal diagnosis and to the prenatal detection of the Y chromosome in amniotic cells seems at least as promising as has proved the earlier identification of the Barr body.

Amniotic Fluid↗
PubMed1991

Interphase nucleolar organiser regions and survival in squamous cell carcinoma of the bronchus: a 10 year follow up study of 138 cases.

BACKGROUND: Good prognostic indicators for patients with squamous cell carcinoma of the lung would help to determine the most appropriate treatment for individual patients. METHODS: A silver colloid technique that shows interphase nucleolar organiser regions (AgNORs) has been applied to representative paraffin sections from 138 cases of squamous cell carcinoma of the bronchus treated by surgical resection of the primary tumour at East Birmingham Hospital in 1977. Of the 138 patients, 23 (17%) were alive 10 years after their operation. RESULTS: The mean (SD) AgNOR count per cell was significantly higher for all grades of malignancy (well differentiated 10.5 (2.6), moderately differentiated 10.7 (3.2), and poorly differentiated 12.7 (4.5)) than for normal pseudostratified columnar epithelium from non-affected areas (2.3 (0.78)). There was a trend for AgNOR counts to be higher in poorly differentiated tumours, but a wide range of AgNOR counts was observed in all histological grades. AgNOR counts did not predict clinical outcome, irrespective of the stage of the disease, and did not relate to DNA ploidy or the percentage of cells in the proliferation phase of the cell cycle. Nine of 47 patients (19%) with tumours classified as DNA diploid and eight of 63 patients (13%) with DNA aneuploid tumours were alive 10 years after operation. Principal component analysis identified the clinicopathological stage of disease as the variable best related to survival. The percentage of patients surviving 10 years was 30% for stage I, 20% for stage II, 10% for stage IIIa, 9% for stage IIIb, and none for stage IV. CONCLUSION: The AgNOR technique is not of prognostic value in postoperative patients with squamous cell carcinoma of the bronchus.

Adult↗
PubMed1993

Detection of aneuploidy in human interphase spermatozoa by fluorescence in situ hybridization (FISH).

Fluorescence in situ hybridization (FISH) with repetitive-sequence DNA probes was performed on human interphase sperm to determine the utility of this technique for aneuploidy detection. DNA sequences specific for chromosomes 15 and 16 and the Y were biotinylated and hybridized with human sperm that had been treated with cetyltrimethylammonium bromide and dithiothreitol to render them accessible to the probes. Fluoresceinated avidin and antiavidin were utilized to visualize bound probe. More than 10,000 sperm were analyzed for each chromosome probe. The hybridization efficiency was 98-99% for all three probes. The frequencies for disomy (i.e., for nuclei containing two fluorescent signals) for chromosomes 15 and 16 and the Y were 0.14%, 0.17%, and 0.11%, respectively. These frequencies are all somewhat higher than those obtained by the human sperm karyotyping technique. Nevertheless, these preliminary results suggest that FISH is a simple and rapid technique that may provide an accurate screen for aneuploidy detection in human sperm.

Aneuploidy↗
PubMed1996

Simultaneous enumeration of chromosomes 13, 18, 21, X, and Y in interphase cells for preimplantation genetic diagnosis of aneuploidy.

A fluorescence in situ hybridization (FISH) protocol to simultaneously enumerate chromosomes 13, 18, 21, X, and Y in interphase cell nuclei for application in preimplantation genetic diagnosis (PGD) of aneuploidy was tested. Strict scoring criteria were developed to minimize recording errors. The protocol used optimized probes for chromosome-specific DNA repeat and single-copy loci and showed a significantly higher efficiency (95%) than previously published protocols. The other purpose of this study was to differentiate between two signals originating from a single split target or from two targets in close proximity. These criteria were based on the FISH results obtained from the analysis of all or most of the cells from 50 chromosomally normal or mosaic human embryos, 20 aneuploid embryos, and five polyploid embryos donated for research. Subsequently, 183 human embryos underwent PGD of aneuploidy in one of their cells. In 64 embryos that were not transferred back to the uterus, the rest of cells were also analyzed, and the previous results were confirmed in 91% of these embryos, with 0% (0/21) of the embryos classified as normal embryos being abnormal and 14% (6/43) of the embryos classified as abnormal being normal. Compared to previous protocols, these criteria minimize the risk of transferring abnormal embryos after PGD analysis.

Aneuploidy↗