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PubMed1978

Kinetic and thermodynamic aspects of in vitro interphase transfer of tetracyclines II: influence of divalent metal salts.

With a two-phase in vitro model composed of an aqueous pH 5.5 buffer and 1-octanol, the kinetics of the interphase transfer of tetracycline derivatives were examined in the presence and absence of calcium and magnesium salts, and the contribution of some functional group substituents to the "apparent" free energy changes for partitioning of tetracyclines was evaluated. Only small changes were observed in kf, kb, K0w, and apparent functional group free energy changes, deltaFG deltaF, in the presence of divalent metals as compared to values observed in the absence of these metals. Introduction of the C-6beta hydroxyl group on the tetracycline nucleus decreased the apparent K0w because of a positive apparent deltaFG deltaF contribution, whereas introduction of C-5 hydroxyl, C-6alpha methyl, or C-7 chloro groups increased the apparent K0w through negative apparent deltaFG deltaF contributions.

Cations, Divalent↗

PubMed1991

Interphase cytogenetics in paraffin sections of routinely processed hydatidiform moles and hydropic abortions.

The differential diagnosis of complete (CM) and partial (PM) hydatidiform moles and hydropic abortions (HA) can be difficult when based on histology alone. Therefore, a more objective approach of chromosome ploidy analysis as detected by in situ hybridization (ISH) was performed on 6 microns paraffin sections of seven cases, originally classified as three CM, two PM, and two HA with a histologic pattern suggestive of triploidy. Probes for repetitive DNA targets in the (peri)centromeric region of chromosomes 1 and X and in the q arm of chromosome Y were used to determine chromosome ploidy and sex chromosome composition. The findings in the three CM were consistent with diploidy: two copies of chromosomes 1 and X and none of chromosome Y. In the two HA with a histologic pattern suggestive of triploidy, three copies of chromosomes 1 and X and none of chromosome Y confirmed triploidy. Two cases originally classified as PM both appeared to have two copies of chromosome 1 with an XX pattern in one case and an XY pattern in the other case, which is consistent with diploidy instead of triploidy. After reviewing, both cases most likely represented CM. We conclude that interphase cytogenetics by ISH on paraffin sections of hydatidiform moles and hydropic abortions enables chromosome ploidy analysis with preservation of histological context.(ABSTRACT TRUNCATED AT 250 WORDS)

Abortion, Missed↗

PubMed2001

Prenatal diagnosis using interphase fluorescence in situ hybridization (FISH): 2-year multi-center retrospective study and review of the literature.

Since 1993, the position of the American College of Medical Genetics (ACMG) has been that prenatal interphase fluorescence in situ hybridization (FISH) is investigational. In 1997, the FDA cleared the AneuVysion assay (Vysis, Inc.) to enumerate chromosomes 13, 18, 21, X and Y for prenatal diagnosis. Data is presented from the clinical trial that led to regulatory clearance (1379 pregnancies) and from retrospective case review on 5197 new pregnancies. These studies demonstrated an extremely high concordance rate between FISH and standard cytogenetics (99.8%) for specific abnormalities that the AneuVysion assay is designed to detect. In 29 039 informative testing events (6576 new and 22 463 cases in the literature) only one false positive (false positive rate = 0.003%) and seven false negative results (false negative rate = 0.024%) occurred. A historical review of all known accounts of specimens tested is presented (29 039 using AneuVysion and 18 275 specimens tested with other probes). These performance characteristics support a prenatal management strategy that includes utilization of FISH for prenatal testing when a diagnosis of aneuploidy of chromosome 13, 18, 21, X or Y is highly suspected by virtue of maternal age, positive maternal serum biochemical screening or abnormal ultrasound findings.

Chromosome Aberrations↗

PubMed1995

The experimental homologous and heterologous separation distance histograms for the centromeres of chromosomes 7, 11, and 17 in interphase human T-lymphocytes.

The simultaneous 3D arrangement of the interphase centromeres of chromosomes 7, 11, and 17 in unstimulated human T-lymphocytes is analyzed. Using triple in situ hybridization in combination with optical sectioning and image processing, the identification of three pairs of centromeres in each nucleus and the assignment of 3D coordinates to each centromere are made. The homologous and heterologous centromere separation distance histograms are determined and compared to the hypothesized histograms for randomly distributed centromeres. The experimental nuclei are truncated spheres in shape with a principal radius of 3.7 +/- 0.3 micron and a truncated hemispherical height of 2.6 micron. None of the separation distance distributions appears to be statistically significantly different from a random model distribution.

Centromere↗

PubMed1985

Mitotic arrest and interphase inhibition induced by the pyrimidine sulfoxide NY 4138.

Phase-specific cell-cycle inhibitory effects of the sulfoxide 5-bromo-2-(2-thienylmethyl)sulfinyl-pyrimidine (NY 4138) was studied on synchronized human NHIK 3025 cells cultivated in vitro. When added to exponentially growing cells, NY 4138 induced an accumulation of metaphases. The metaphase arrest was, however, not efficient immediately after addition of the compound. While a few telophases were still present 3 h after addition of 0.1 mM, no telophases were seen 6 h after addition. Most cells exposed to metaphase arrest by NY 4138 lost their ability to form colonies. We therefore characterize the metaphase arrest induced by this drug as irreversible. For concentrations above 0.5 mM phase specific cell cycle inhibition was also observed during interphase. Cells treated in G1 only were not delayed in G1 but were delayed in S and in G2. Cells treated in S only were also delayed in S, but to a smaller extent than the cells treated in G1 only. Cells treated with 0.1 mM NY 4138 for a period of 4 h in either G1 or in S showed no significant prolongation of the subsequent mitosis (i.e. the drug was in this case not present during mitosis). However, about 20% of the cells in this case divided to form 3 daughter cells of which 1 had a normal G1 DNA-content, 1 had 1/3 and 1 had 2/3 of a normal G1 DNA-content.

Cell Division↗

PubMed1979

Localisation of nucleolus-organizing regions in interphase cells.

A technique based on the use of silver solutions, which selectively stains the nucleolus-organising regions (NORs) in chromosomes, was applied to interphase Ehrlich tumour cells. The results indicate that nucleolar fibrillar centres correspond to the NORs.

Animals↗

PubMed1975

The organization of interphase chromatin in drosophilidae: the self adhesion of chromatin containing the same DNA sequences.

Cytological evidence is presented which shows that for Drosophila virilis and Samoaia leonensis at least, each satellite DNA is condensed into a distinct heterochromatic mass during interphase. This is seen as just one example of a general phenomenon in which chromatin containing a particular DNA sequence binds to other chromatin containing the same sequence. It is proposed that DNA sequence specific proteins can account for this phenomenon.

Animals↗

PubMed1985

Protein composition of the chromosomal scaffold and interphase nuclear matrix.

Residual protein structures were prepared from isolated chromosomes and interphase nuclei of in vitro cultured bovine liver cells and the protein compositions were analysed. Chromosomes with minimal cytoplasmic contamination were obtained by a simple procedure using a pH 8 isolation medium containing Triton X-100 and polyamines, and residual protein-DNA complexes were prepared by extraction with 2 M NaCl. Residual protein structures were also obtained by digesting isolated chromosomes with staphylococcal nuclease. Protein compositions of both structures as obtained by SDS-polyacrylamide gel electrophoresis were essentially the same. Residual protein structures were prepared from isolated nuclei by the same procedures. The major nuclear matrix proteins, i.e., the lamins A, B, and C, were not found in the chromosomes and chromosome scaffolds. On the other hand, the residual chromosome structures contained two major polypeptides of 37 and 83 kilodalton relative molecular weights that were absent from the nuclear matrix preparations. A few polypeptides with the same or very similar electrophoretic mobilities were found in the residual structures of both the nuclei and the chromosomes.

Animals↗

PubMed1976

Occurrence of paired cisternae in the cytoplasm of interphase murine mammary carcinoma cells.

The presence of paired cisternae in the cytoplasm interphase murine mammary carcinoma cells is described. The paired cisternae are composed of about 65A thin trilaminated membranes, about 65A in thickness, with ribosomes attached to the outer membranes, but with smooth adjacent membranes between the cisternae. The function and origin of these structures were not clarified, but it is suggested that they represent surplus fragments of nuclear envelope material.

Animals↗

PubMed1978

Methods of denaturation and renaturation of DNA in interphasic chromatin: cytochemical quantitative analysis by Methyl Green staining.

Almost diploid nuclei (as judged from the microdensitometric evaluation of the Feulgen positive material) of granular and Purkinje cells of the rat cerebellar cortex, were submitted to in situ DNA denaturation and renaturation experiments. We assessed the double-strandedness of DNA, by Methyl Green staining according to Scott (1967). Under these conditions a stoichiometric ratio between bound dye and DNA exists, suitable for quantitative microdensitometric measurements. Our data show that DNA in the interphasic chromatin is never completely denatured after the treatments we used. Furthermore, the renaturation takes place in a different way in the two cell types. Owing to the unlike chromatin packing of granular and Purkinje nuclei, we suggest that nuclear proteins must interfere differently on the in situ denaturation and renaturation processes.

Animals↗

PubMed1993

Detection of confined placental mosaicism in trisomy 18 conceptions using interphase cytogenetic analysis.

Fluorescence in situ hybridization provides a rapid and accurate technique for detecting chromosomal aneuploidy. It is an excellent method for identifying mosaicism in placental tissues following prenatal diagnosis. Mosaicism, in the form of confined placental mosaicism, occurs im approximately 1%-2% of viable pregnancies studied by chorionic villus sampling at 9-11 weeks of gestation. It has been detected in pregnancies with both diploid and trisomic fetuses and appears to have an important effect on the intrauterine fetal survival. Using both standard cytogenetic analysis and fluorescence in situ hybridization, we have studied 12 placentas from pregnancies with trisomy 18 for the presence of chromosomal mosaicism. These included 2 that were spontaneously aborted, 5 that were terminated after prenatal diagnosis, and 4 that were delivered as either stillborn or liveborn. Significant levels of mosaicism, confined exclusively to cytotrophoblast, were detected in 7 pregnancies. This study demonstrates the usefulness of interphase cytogenetic analysis of uncultured tissues as an alternative method for the detection of mosaicism.

Aneuploidy↗

PubMed1988

Interphasic nucleolar organizer region distribution as a diagnostic parameter to differentiate benign from malignant epithelial tumors of human intestine.

The distribution of the interphasic nucleolar organizer regions (NORs) has been investigated in five hyperplastic polyps, five adenomatous polyps and fifteen colonic adenocarcinomas. The study was performed using electron microscopy and paraffin-embedded sections stained for Ag-NOR proteins. Malignant tumor cells were characterized by a large number of NORs which were small in size and showed a scattered distribution. Nuclei of both types of polyp had only a small number of large-sized NORs in a clustered distribution. In two adenomatous polyps, cells were also observed with an NOR distribution pattern intermediate between that of frankly benign and malignant lesions.

Adenocarcinoma↗

PubMed1992

Interphasic nucleolar organizer regions expression and cell kinetics evaluation during gastric carcinogenesis induced by nitrosoguanidine in the rat.

An increased number of interphasic nucleolar organizer regions containing ribosomal cistrons associated with argyrophilic proteins (AgNORs) has been described in human malignant tumor cells. In this study variations in AgNOR numbers have been compared with changes of cell kinetics, evaluated by the mitotic count (MC) and bromodeoxyuridine labeling index (BrdU LI), during gastric carcinogenesis induced with N-methyl-N'-nitro-N-nitrosoguanidine (NG) in rats. Significant differences (2 P < 0.005) in AgNOR mean numbers, evaluated in the antral isthmic cells, in MC mean values and BrdU LI, evaluated in the whole antral cellular population, were found when comparing areas of acute gastritis, atrophy and hyperplasia in NG-treated rats with the normal mucosa in controls. No differences were observed in MC and BrdU LI between normal antrum and carcinoma cells which showed an AgNORs mean number lower than in the isthmic cells of controls (2 P < 0.005). Moreover, significant correlations were found comparing changes in AgNOR numbers with MC (r = 0.89, P < 0.001) and BrdU LI (r = 0.66, P < 0.001) in different lesions. These data show that evaluation of AgNOR numbers does not allow the identification of malignant cells in NG-induced gastric carcinoma. However AgNOR quantification seems to be a reliable index of cell kinetics and related well with the cellular dividing fraction.

Adenocarcinoma↗

PubMed2002

Detection of the BCR-ABL gene by interphase fluorescence in situ hybridization (iFISH) in chronic myelogenous leukemia patients after hemopoietic stem cell transplantation: the feasibility of iFISH monitoring of therapeutic response in peripheral blood.

The detection of the Philadelphia (Ph) translocation has been accomplished primarily by cytogenetic analysis and reverse transcriptase polymerase chain reaction (RT-PCR). RT-PCR is highly sensitive (1/10(4)-10(6)) but not quantitatively reliable and is thus unsuitable for the monitoring of Ph-positive cells during therapy. Interphase fluorescence in situ hybridization (iFISH) allows analysis of a large number of cells (> 500) in a timely and efficiently quantitative manner. We obtained 118 peripheral blood (PB) and 127 bone marrow (BM) samples from 75 adult chronic myelogenous leukemia (CML) patients undergoing stem cell transplantation. We simultaneously performed nested RT-PCR and iFISH for all samples. False-positive cells were detected in 2.48% +/- 0.93% (mean +/- SD) of PB samples and 2.75% +/- 0.83% of BM samples. The iFISH results for PB and BM ranged from 1.4% to 92.8% and 1.0% to 93.8%, respectively. Correlation analysis of iFISH results for PB versus BM samples showed a strong relation (r = .993). A significant correlation (P < .05) was also found between iFISH and first-round RT-PCR. The sensitivity of BCR-ABL iFISH was similar to that of first-round RT-PCR, and iFISH results for PB and BM were also well correlated. Thus, iFISH analysis of PB and/or BM samples may be more clinically reliable than RT-PCR in the quantitative monitoring of BCR-ABL fusion in CML after transplantation.

Blood Cells↗

PubMed2001

Multiple chromosomal underrepresentations detected by interphase cytogenetics - possible prognostic markers in head and neck tumors?

Relevant prognostic factors for head and neck squamous cell carcinoma are tumor extension (pT), occurrence of lymph node metastases (pN) and grade of differentiation (G). We tried to correlate these histological characteristics with numerical aberrations of whole chromosomes as demonstrated by fluorescence in situ hybridization techniques (FISH). Therefore, we investigated isolated interphase cells from paraffin sections of squamous cell carcinomas of the head and neck region from 46 patients with centromeric DNA probes for chromosomes 1, 3, 4, 6, 7, 9, 10, 11, 12, 15, 17, 18, X and Y. The majority of tumor samples showed aneuploidy for most chromosomes analyzed. The main chromosomal abnormality was loss of chromosomal material, predominantly of chromosomes 3 (28%), 6 (20%), 9 (26%), 10 (24%) and 18 (33%). Multiple deletions could be demonstrated more frequently in poorly differentiated carcinomas (88% G3-tumors with more than one deletion in contrast to 66% G2-tumors). The occurrence of multiple deletions may also correlate with progression in lymph node metastasis (66% in pN0-tumors vs. 85% in pN2-tumors), whereas the differences between the stages of primary tumor extension were not so obvious. Despite of a some-what disproportionate distribution of tumors in the different pT- and pN-stages and the rather low number of cases, our results suggest a relationship between the quantity of chromosomal underrepresentation, grade of differentiation and higher lymph node stage. Therefore, they underline the importance of chromosomal deletions as a possible additional prognostic marker in head and neck squamous cell carcinoma.

Aneuploidy↗

PubMed1981

Uneven distribution of palmitoyl carnitine in solutions because of migration to air/water interphase.

Standard solutions of palmitoyl carnitine could not be prepared in water because, even at below critical micelle concentrations, palmitoyl carnitine did not distribute uniformly in solutions. Evidence indicates that palmitoyl carnitine prefers to leave the bulk phase to segregate readily at the water/air and water/apolar interphases. Thus, in metabolic and kinetic studies, the actual concentration of long-chain acyl carnitines available for reactions at any instant can be drastically different from that calculated from the amounts added.

Air↗

PubMed1993

Pentobarbital, diazepam, and ethanol abolish the interphase diminution of pain in the formalin test: evidence for pain modulation by GABAA receptors.

There are two phases to the behavioral response to injection of formalin. After an initial vigorous response, a period of reduced pain occurs 10 to 15 min after formalin, followed by reemergence of pain-related behaviors. These phases are believed to represent acute chemical stimulation of afferent neurons followed by injury-related inflammatory pain. Pentobarbital (10, 15, or 25 mg/kg), diazepam (0.5, 1.5, or 5.0 mg/kg), or ethanol (0.5, 1.0, or 1.5 g/kg) attenuated the diminution of pain between the two phases, so that pain was continuous throughout 60 min of testing, but had no effect on pain scores during the peaks of either phase. The effects of pentobarbital and diazepam were blocked by picrotoxin (2.5 mg/kg), which itself had no effect. Ro 15-1788 also blocked the effect of diazepam. Picrotoxin did not effectively antagonize the effect of ethanol. A high dose of picrotoxin (5.0 mg/kg) caused seizures in some rats and also eliminated the interphase depression of pain. The results suggest that the biphasic time course of formalin pain is produced by a central antinociceptive mechanism that is inhibited by GABAA receptors.

Analgesics↗

PubMed1976

Isolation, characterization, and structure of the folded interphase genome of Drosophila melanogaster.

The intact interphase genome of Drosophila melanogaster has been isolated by sucrose gradient centrifugation after gentle lysis of tissue culture cells in 0.9 M NaCl-0.4% nonidet P40. The non-viscous folded DNA sediments as a single broad 5000S peak in a complex with RNA (a fraction of the nuclear nascent RNA) and protein (all of the four intranuclesome histones: H2A, H2B, H3, and H4). The folded DNA is supercoiled and can be relaxed to slower sedimenting forms either by intercalating ethidium or by nicking with DNAase I. Incomplete DNAase treatment gives partially relaxed complexes, indicating that each nick relaxes only a stretch of DNA (defined as a supercoiled DNA loop) without affecting the superhelical content of the rest of the genome. The concentration of superhelices in the Drosophila folded DNA is the same as in the E. coli and SV40 closed circular DNAs-that is, about one negative turn every 200 base pairs (bp) in 0.15 M NaCl at 26 degrees C. The estimated average size of the supercoiled DNA loops, about 85,000 bp, equals the size of the larger Drosophila chromomeres. Ethidium intercalation in 0.9 M NaCl both removes the negative superhelical turns and dissociates the four histones from the DNA. The four histones are dissociated in equimolar concentrations, and the relative proportion of histones displaced from the DNA is a function of ethidium concentration. The histones are completely dissociated from the folded DNA at the ethidium concentration. The histones are completely dissociated from the folded DNA at the ethidium concentration which removes all of the negative superhelices. Thus the data strongly suggest that the rotation of the Watson Crick helix which accompanies ethidium intercalation causes the loss of nucleosomes from the DNA. The results are interpreted in terms of a model for the folded Drosophila genome which has the DNA constrained (by both protein-DNA and RNA- DNA interactions) into independent supercoiled loops containing on the average 400 nucleosomes per loop. Each nucleosome is composed of a histone core with the DNA wound around it in a 360 degrees left-handed toroidal supercoil; each nucleosome toroidal supercoil plus its relaxed internucleosome DNA contains, on the average, 200 bp.

Animals↗