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PubMed1990

Interphase nuclear envelope lamins form a discontinuous network that interacts with only a fraction of the chromatin in the nuclear periphery.

Antibodies directed against nuclear envelope lamin proteins have been used in conjunction with three-dimensional light and electron microscope methodologies to determine the spatial organization of lamins in diploid interphase nuclei and to relate this organization to the positions of chromatin in the nuclear periphery. Using Drosophila early embryos, Drosophila Kc cells, and human HeLa cells, it is qualitatively and quantitatively observed that lamins are organized as a highly discontinuous, apparently fibrillar network that leaves large voids in the nuclear periphery containing little or no lamin. Using fluorescence microscopy to compare and quantitate the relationship between chromatin and the lamin network, it is found that although there is a strong tendency for the most peripheral chromatin to be positioned directly underneath a lamin fiber, only a small fraction of the chromatin in the nuclear periphery is sufficiently close to a lamin fiber to possibly be in direct contact.

Animals↗

PubMed1981

An interphase model for mitotic chromosome organization in eukaryota.

A model for the spatial relationship of the arrangement of the chromosomes in the nucleus in eukaryota is presented. Evidence is derived from light and electron microscopic studies, application of autoradiographic and banding techniques; on the organization, structure and behaviour of chromosomes during interphase and other stages of cell cycle. This model visualizes the entire chromosomal DNA as a single uninemic multirepliconic continuum where the chromosomes with their centromeres and telomeres have a predetermined arrangement among themselves as well as in relation to the nucleolus and nuclear membrane. This orderly arrangement is presumably maintained through interchromosomal connections. The impact of this model on the interpretation of various cytogenetic phenomena is discussed.

Animals↗

PubMed1985

Response of interphasic nucleoli to hypoxia in root meristems.

Hypoxia produces structural changes in interphasic nucleoli of Allium cepa L. root meristems. Following segregation, the fibrillar portion of nucleoli seems to be extruded in masses, accessory bodies, which stay in nucleoplasm. Such bodies apparently leave the fibrillar portion in places which seem to correspond to insertion areas of the nucleolar organizers in nucleoli. Reversal of these changes is rapidly obtained when oxygenation is restored. Accessory bodies differ from prenucleolar bodies since they are compact and homogeneous and they do not display segregation under hypoxia.

Cell Nucleolus↗

PubMed1996

1H MAS NMR spectroscopy of chemically modified silica gels: a fast method to characterize stationary interphases for chromatography.

Chemically modified silica gels used as stationary phases in chromatography have been investigated by means of solid-state 1H magic angle spinning (MAS) NMR spectroscopy. Since the organosilanes are bonded to the surface of the silica gel, their protons are diluted and possess a higher mobility in comparison to protons in pure organic solids. Thereby the usually strong homonuclear dipole-dipole interactions among the protons are reduced and it is possible to obtain well-resolved 1H NMR spectra of the organic interphases with MAS-only techniques. Effects of temperature and magnetic field strength on the resolution of the spectra are examined as well as the dependence of T1 and T1pH relaxation times on temperature and spinning speed.

Chemical Phenomena↗

PubMed2004

Specificity of interphase fluorescence in situ hybridization for detection of chromosome aberrations in tumor pathology.

Interphase fluorescence in situ hybridization (IFISH) is an interesting and intriguing cytogenetic approach in the study of tumor chromosomal abnormalities when metaphases are not available. This technique can be applied to different types of tumor nuclei, including imprinted nuclei (IM), nuclei obtained from conventional cytogenetic procedures (PB), frozen nuclei, paraffin-embedded nuclei (PE), and nuclei extracted from paraffin-embedded sections (EX). IFISH is a high-sensitivity approach in tumor studies that can give evidence of genetic aberrations present in a small percentage of cells that are likely to escape detection if only molecular techniques are applied. Despite its high sensitivity and versatility, IFISH is an indirect cytogenetic method and needs controls to have adequate specificity. This study includes present data obtained in IFISH experiments using different types of probes (alpha-satellite and YAC clones) hybridized on different types of normal control nuclei, such as PB, IM, EX, and PE nuclei, to define the threshold level for monosomy and trisomy of different chromosomal regions. My findings demonstrate that the cut-off values depend both on the types of probes and on the types of target nuclei. Therefore, even if IFISH is a versatile, high-sensitivity technique for detecting chromosomal abnormalities, the lack of accurate controls may result in the misdiagnosis of some abnormalities.

Bone Marrow↗

PubMed2006

Effect of interphase gap and pulse duration on electrically evoked potentials is correlated with auditory nerve survival.

We investigated the effect of pulse duration (PD) and interphase-gap (IPG) on the electrically-evoked auditory brain stem response (EABR) and viiith nerve compound action potential (ECAP) of deafened guinea pigs in order to test the hypothesis that the extent of change in these neural responses is affected by the histological status of the auditory nerve. Fifteen guinea pigs were deafened by co-administration of kanamycin and furosemide. Animals were acutely implanted with an 8-band electrode array at 1, 4 or 12 weeks following deafening. EABR and ECAP input/output functions were recorded in response to charge balanced biphasic current pulses. We determined the change in current required to equalize; (i) the EABR amplitude when the duration of the current pulse was doubled (104-208 micros/phase); and (ii) the EABR and ECAP amplitudes when the IPG was increased from 8 to 58 micros using a 104 micros/phase current pulse. Following the completion of each experiment the cochleae were examined quantitatively for spiral ganglion neuron survival. As expected, the current level required to evoke an EABR with equal amplitude was lower when the animal was stimulated with current pulses of 208 compared with 104 micros/phase. Moreover, the current level required to evoke EABR/ECAPs with equal amplitude was lower when current pulses had an IPG of 58 versus 8 micros. Importantly, there was a reduction in the magnitude of this effect with greater neural loss; the reduced efficacy of changing both PD and IPG on these electrically-evoked potentials was statistically correlated with neural survival. These results may provide a tool for investigating the contribution of auditory nerve survival to clinical performance among cochlear implant subjects.

Action Potentials↗

PubMed2006

Quantitation of chromosome 1p and 19q deletions in glial tumours by interphase FISH on formalin-fixed paraffin-embedded tissue.

Deletions on chromosomes 1p and 19q identify oligodendroglial tumours that are likely to have a complete response to some chemotherapy regimens and are associated with prolonged patient survival. Detection of these chromosomal alterations is becoming increasingly important in the evaluation of glial tumours. Interphase fluorescence in situ hybridisation (FISH) is one of a number of techniques for detecting deletions, and is an efficient method for screening large numbers of tumours. We used FISH to detect 1p and 19q deletions in formalin-fixed paraffin sections of 11 oligodendrogliomas, 11 oligoastrocytomas, two astrocytomas and four glioblastomas multiforme. 1p and 19q deletion and non-deletion ratios were tabulated in 200 nuclei in each tumour. We found considerable variation between tumours in the fraction of cells with deletions. This variation has not been reported previously and its clinical significance will be clarified with patient follow-up. There was little variation between regions within the same tumour.

Brain Neoplasms↗

PubMed1975

Stimulation of late interphase Dictyostelium discoideum amoebae with an external cyclic AMP signal.

The microelectrode system described in the accompanying paper was used to investigate properties of fields of Dictyostelium discoideum amoebae in late interphase. Cells in the fields were competent to respond chemotactically to, and to relay, a c-AMP signal, but not to produce an aggregative signal autonomously. The experimental results are generally consistent with c-AMP being the sole compound required for chemotaxis and signal relaying. A periodic signal from the microelectrode can initiate and control aggregation and can complete with spontaneously arising aggregates. The electrode was used to measure the refractory period for relaying which decreases from 9 min or more to between 2 and 3 min with increasing developmental age, and to measure thresholds for chemotaxis and signal relaying. The results are discussed in relation to models for the control of aggregation in D. discoideum.

Cyclic AMP↗

PubMed1983

Conformation of glucagon in a lipid-water interphase by 1H nuclear magnetic resonance.

A determination of the spatial structure of the polypeptide hormone glucagon bound to perdeuterated dodecylphosphocholine micelles is described. A map of distance constraints between individually assigned hydrogen atoms of the polypeptide chain was obtained from two-dimensional nuclear Overhauser enhancement spectroscopy. These data were used as the input for a distance geometry algorithm for computing conformations that would be compatible with the experiments. In the region from residues 5 to 29 the mobility of the polypeptide backbone and most of the amino acid side-chains was found to be essentially restricted to the overall rotational tumbling of the micelles. The secondary structure in this region includes three turns of irregular alpha-helix in the segment of residues 17 to 29 near the C terminus, a stretch of extended polypeptide chain from residues 14 to 17, an alpha-helix-like turn formed by the residues 10 to 14 and another extended region from residues 5 to 10. In the N-terminal tetrapeptide H-His-Ser-Gln-Gly- the two terminal residues are highly mobile, indicating that they extend into the aqueous phase, and the mobility of the residues Gln3 and Gly4 appears to be only partially restricted by the binding to the micelle. The absence of long range nuclear Overhauser effects between the peptide segments 5-9 and 11-29, and between 5-16 and 19-29 shows that the polypeptide chain does not fold back on itself and hence that micelle-bound glucagon does not adopt a globular tertiary structure. Previously it was shown that the polypeptide backbone of glucagon is located close to and runs roughly parallel to the micelle surface. Combination of these observations suggests that the overall spatial arrangement of the glucagon polypeptide chain in a lipid-water interphase is largely determined by the topology of the lipid support, in the present case the curvature of the dodecylphosphocholine micelles. The tertiary structure is further characterized by the formation of two hydrophobic patches by the side-chains of Phe6, Tyr10 and Leu14, and the side-chains of Ala19, Phe22, Val23, Trp25 and Leu26, respectively.

Amino Acid Sequence↗

PubMed2001

Distal deletion of 1p in colorectal tumors: an initial event and/or a step in carcinogenesis? Study by fluorescence in situ hybridization interphase cytogenetics.

Cytogenetics studies have suggested that short arm deletion in chromosome 1 is involved in triggering colorectal tumor development. To elucidate the role of 1p under-representation in the tumoral process, we investigated by fluorescence in situ hybridization interphase cytogenetics, using simultaneously centromeric and p36 telomeric probes for chromosome 1, 27 primary adenocarcinomas, 5 metastases, 5 adenomas and as control 4 normal mucous membranes. The 1p under-representation in paradiploid tumoral cells, interpreted as a 1p deletion, was observed in 8/27 adenocarcinomas, 2/5 metastases and 3/5 adenomas. Thus, in diploid cells 1p deletion was observed in some tumors independently of the stage of the process. The 1p under-representation in total number of examined cells, i.e., diploid and aneuploid, was observed in 14/16 grade B1-B2 tumors, in 5/8 grade C1-C2 tumors, and all grade D tumors (3/3) and all metastases (5/5). There were no correlations with location or histological characteristics of cancers, gender or age of patients. These results show high frequency of 1p under-representation in intestinal tumors, and lead to separate the under-representation of 1p in diploid cells, which correspond to a 1p deletion probably implicated in the initiation of the process, from the under-representation in aneuploid cells, which mainly may be the consequence of complex rearrangements in relation to extension of the malignant process.

Adenocarcinoma↗

PubMed2002

Interphase fluorescence in situ hybridization and DNA flow cytometry analysis of medulloblastomas with a normal karyotype.

Interphase fluorescence in situ hybridization (FISH) with chromosome 3 and 17 centromeric probes and DNA flow cytometry were used for a retrospective study of nine pediatric medulloblastomas with normal karyotypes after tissue culture. The FISH analysis of medulloblastoma touch preparations showed that in seven of nine tumors, a significant proportion of nuclei had an increased number of centromeric signals for the selected chromosomes. In six of seven cases, this increase was caused by the presence of triploid and tetraploid clones as established by flow cytometry of paraffin-embedded tumors. These findings show that molecular cytogenetic analysis combined with DNA flow cytometry is necessary for all pediatric medulloblastomas diagnosed as cytogenetically normal on cultured tumor tissue.

Cerebellar Neoplasms↗

PubMed1995

Detection of 9p deletions in leukemia cell lines by interphase fluorescence in situ hybridization with YAC-derived probes.

Hemizygous and homozygous deletions of the type I interferon gene cluster (IFN) have been detected in about 20% of acute lymphoblastic leukemias. A putative tumor suppressor gene (TSG) is thought to be located centromeric to the IFN cluster on chromosomal bands 9p21-22. We studied the accuracy of fluorescence in situ hybridization (FISH) for detecting deletions in interphase cells using yeast artificial chromosome (YAC) clones containing all or part of the IFN cluster. FISH probes were generated from YACs (320-1300 kb in size) by a sequence-independent amplification technique (SIA). Fifteen cell lines (nine T-ALL, three B-cell precursor ALL, one B-ALL, one AML, one CML-BC) that had been well characterized by conventional cytogenetic analysis and molecular techniques were analyzed. We were able to detect all numerical changes of the IFN cluster including homozygous and hemizygous deletions accurately and to define subclones of the cell lines. Moreover, in six cell lines we were able to identify subclones. In dilution experiments the detection thresholds for subpopulations with homozygous and hemizygous deletions were determined to be 5% and 7.5%, respectively.

Base Sequence↗

PubMed1996

Tetrasomy 8 detected by interphase cytogenetics in a child with acute lymphocytic leukemia.

Tetrasomy 8 is a rare clonal anomaly in human acute leukemia. Here we present a case of a 7-year-old boy with acute lymphoblastic leukemia (ALL) displaying a tetrasomy 8 clone that could not be detected by conventional cytogenetics. In this study, bone marrow and peripheral blood cells were collected at five different diagnostic stages and analyzed by double targeted fluorescence in situ hybridization (FISH) with centromeric DNA probes for chromosomes 7, 8, 9, and 12. FISH analysis revealed a significant increase in tetrasomy 8 frequency, but not in other chromosomes examined. A smaller increase in trisomy 8 was also detected. At one stage over 60% of the cells were hyperdiploid with 40% being tetrasomic. The size of the tetrasomic clone changed during the course of the disease. The hyperdiploid frequencies of chromosome 8 detected by interphase FISH analysis in bone marrow and peripheral blood were similar. Our findings indicate the utility of FISH analysis in cytogenetic monitoring of leukemia patients and further show that tetrasomy 8 may play a specific role in a subtype of ALL.

Aneuploidy↗

PubMed1997

Ph-positive CML in blastic phase with monosomy 7 in a Down syndrome patient. Monitoring by interphase cytogenetics and demonstration of maternal allelic loss.

We report a case of Ph-positive chronic myelocytic leukemia in blastic phase in an 11-year-old boy with Down syndrome. Monosomy 7 was the only additional chromosomal anomaly in the blastic clone. Fluorescence in situ hybridization analysis on interphase nuclei with a centromeric probe specific to chromosome 7 proved to be efficient in disease monitoring, and showed, together with the results of chromosome analysis on metaphases, that B-lymphocytes at the origin of an EBV-established line were not part of the leukemic clone. The study of DNA polymorphisms showed that the origin of the constitutional trisomy 21 was a maternal anaphase I nondisjunction, that the chromosome 7 lost in the blastic marrow clone was the maternal one, and led us to postulate that the mother's chromosomes are prone to impairment of normal disjunction. The study of allelic losses of chromosome 7 loci proved to be a further possibility for disease monitoring.

Adult↗

PubMed1998

The value of interphase fluorescence in situ hybridization in the study of patients with lymphoproliferative disorders: further evidence for a higher sensitivity of detecting chromosomes 7 and 8 aneuploidy.

Dual-color interphase fluorescence in situ hybridization (I-FISH) for chromosomes 7 and 8 was studied retrospectively on 32 patients with suspected lymphoid disorders, and the results were compared with standard cytogenetics. One of 29 (3.4%) patients with lymphoid malignancy showed cytogenetically detectable aneuploidy for chromosomes 7 and 8. In an additional 5 patients (17.2%), I-FISH unmasked chromosomal loss and gain that were not detected by standard metaphase analysis. This represents 19% of the 21 studied patients with acute lymphoblastic leukemia (ALL). These findings indicate that aneuploidies for chromosomes 7 and 8 are underreported in ALL and further demonstrate higher sensitivity of I-FISH for detecting numerical chromosomal rearrangements in leukemic cells.

Adult↗

PubMed1998

Concurrent cytogenetic, interphase fluorescence in situ hybridization and DNA flow cytometric analyses of a carcinoma ex-pleomorphic adenoma of parotid gland.

We report the cytogenetic, fluorescence in situ hybridization (FISH), and DNA ploidy analyses of a high grade carcinoma ex-pleomorphic adenoma of the submandibular gland. Our overall combined analyses showed a marked DNA aneuploidy and numerical abnormalities involving all chromosomes. Cytogenetic analysis revealed a near tetraploid modal chromosomal number with tetraploid loss of chromosomes Y, 1, 6, 9, 11, 14, 15, 17, and 19-21 and hypertetraploid gain of chromosomes 7, 8, and 22. The structural abnormalities included der(1;14)(q10;q10), del(6)(q15q34), +del(6)(q15q34), +der(8) t(1;8)(q12;q12.2),der(9;19)(q10;q10),add(14)(p11.2),i(20)(q10),der(21) t(8;21)(q11.2;q22.3),+der(21)t(8;21) (q11.2;q22.3). Interphase FISH of the primary and short-term cultured cells using directly labeled pericentromeric probes for chromosomes 6-12, 17, 18, and Y resulted in alterations corresponding to the cytogenetic findings. DNA ploidy analysis of both the primary and cultured tumor cells showed a hyperdiploid stemline with DNA indices of 2.6. The results indicate that: (1) marked numerical, structural chromosomal, and DNA content abnormalities are present in this tumor; and (2) alteration at 8q and 6q regions, together with previous results, suggest an association between these events and the development and/or progression of this tumor.

Adenocarcinoma↗

PubMed2000

Cytogenetic and interphase FISH analyses of 73 basal cell and three squamous cell carcinomas: different findings in direct preparations and short-term cell cultures.

Cytogenetic analysis performed on 73 sporadic basal cell carcinomas (BCCs) and three squamous cell carcinomas (SCCs) showed different findings in direct preparations (24 hours) and in short-term cell cultures. Except for loss of the Y chromosome, not one of the other clonal (+6, +16, add(2)(q37), del(3)(q13), add(1)(p31), and near triploidy) or sporadic changes found in direct preparations was found in cell cultures and vice versa. Clonal trisomy 6 found in two BCC direct preparations and demonstrated by interphase fluorescence in situ hybridization in 8 other cases seems to be a nonrandom change in basal cell carcinoma. Immunohistochemistry showed that the cell type investigated was different in the two methods of analysis used: epithelial in direct preparations and fibroblastic in cell cultures. Thus, the results obtained in direct preparations indicate the BCC or SCC epithelial karyotype, whereas the aberrations found in cell cultures indicate the presence of chromosome instability in the fibroblastic stroma. The apparent lack of correspondence between direct and indirect preparations and the presence of clonal chromosome changes in both epithelial and stromal cells suggest tumor cell heterogeneity of BCC. The fibroblastic stroma seems to be implicated in the neoplastic process. This is not evident in SCC, in which clonal changes are present only in direct preparations. The chromosomal distribution of the breakpoints involved in structural changes in direct and cell culture preparations is random; together with those reported in the literature, the breakpoints found in BCC cultures show, however, a cluster to 1p36, 3q13, 9q22, 14p11, 15p11, and Xp11 bands. We did not find any significant correlations between BCC cytogenetic results and the clinical data (site, age, sex, recurrence). The incidence of cases of BCC (38%) and of SCC (100%) showing clonal chromosome changes agree with their benign and malignant nature, respectively. Finally, a significantly high incidence of constitutional inv(9) and dup(9)(q11q21) was found in the group of patients with BCC.

Abdominal Neoplasms↗

PubMed2000

Interphase FISH for Y chromosome, VNTR polymorphisms, and RT-PCR for BCR-ABL in the monitoring of HLA-matched and mismatched transplants.

Thirty-six sex-mismatched transplants were studied using fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR) methods. Molecular cytogenetics was performed using interphase FISH with a centromeric probe for chromosome Y, and PCR amplification was performed with a set of VNTR microsatellite loci. In addition, reverse transcriptase-PCR (RT-PCR) for BCR-ABL fusion was used to investigate cases of Philadelphia chromosome (Ph)-positive chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL). Our integrated approach of post-transplant monitoring was helpful in documenting successful transplants and in controlling the size of Ph-positive clones in CML. A striking overlap was found between results from FISH analysis and PCR for polymorphic loci.

Adolescent↗