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PubMed2001

Diphosphorylated MRLC is required for organization of stress fibers in interphase cells and the contractile ring in dividing cells.

Activity of nonmuscle myosin II is regulated by phosphorylation of its regulatory light chain (MRLC). Phosphoryration of MRLC at both Thr18 and Ser19 (diphosphorylation) results in higher MgATPase activity and in promotion of the assembly of myosin II filaments than does that of MRLC at Ser19 (monophosphorylation) in vitro. To determine the roles of the diphosphorylated MRLC in vivo, we transfected three kinds of MRLC mutants, unphosphorylated, monophosphorylated and diphosphorylated forms (MRLC2(T18AS19A), substitution of both Ser19 and Thr18 by Ala; MRLC2(T18AS19D), Ser19 by Asp and Thr18 by Ala; and MRLC2(T18DS19D), both Ser19 and Thr18 by Asp, respectively), into HeLa cells. Cells overexpressing the mutant MRLC2(T18DS19D) contained a larger number of actin filament bundles than did those overexpressing the mutant MRLC2(T18AS19D). Moreover, cells overexpressing the nonphosphorylatable mutant MRLC2(T18AS19A) showed a decrease in the number of actin filament bundles. Taken together, our data suggest that diphosphorylation of MRLC plays an important role in regulating actin filament assembly and reorganization in nonmuscle cells.

Amino Acid Sequence↗
PubMed1999

Chromosome 8 copy numbers and the c-myc gene amplification in non-small cell lung cancer. Analysis by interphase cytogenetics.

Amplification of the c-myc gene has been reported in non-small cell lung cancer (NSCLC). We performed dual color fluorescence in situ hybridization (FISH) to detect amplifications of the c-myc gene on chromosome 8 to evaluate the relationship between these possible abnormalities and pathological stage. Tumor tissue samples were obtained from 29 patients of NSCLC in Stage I (n = 15) and III (n = 14) who underwent lobectomy at Saitama Cancer Center. Samples were analyzed for chromosome 8 centromere and c-myc gene by dual color FISH. The numerical aberration rate of chromosome 8 was 36.8 +/- 20.3% in Stage I and 40.6 +/- 24.8% in Stage III. The amplification rate of c-myc gene was 48.3 +/- 15.2% in Stage I and 57.4 +/- 17.0% in Stage III. There was a significnat difference in the numerical aberration rate of chromosome 8 between patients who survived for 5 years or more (28.8 +/- 17.5%) and those who survived less than 5 years (44.7 +/- 23.1%). The amplification rate of c-myc gene was not different between patients who survived more and less than 5 years survival, and who survived more and less than 3 years. The 5 year-survival rate in patients who showed 40% or more of chromosome 8 aberrations (n = 13) was 15.4%, which revealed significantly less than that of patients who showed less than 40% of aberrations (n = 16) (56.3%). There was no difference between the 5 year-survival rate in patients whose amplification rates of c-myc gene were equal or more than 50% (n = 16) and less than 50% (n = 13) (25.0% and 53.9%). The rate of chromosome 8 aberrations and the c-myc gene amplification rate were not correlated with pathological stage. However, the rate of chromosome 8 aberration showed correlation in terms of longevity of survival rate, therefore we considered the rate of chromosome 8 aberration to be an additional prognostic factor of patient with NSCLC.

Adult↗
PubMed2005

Rapid prenatal diagnostics in the interphase nucleus: procedure and cut-off rates.

Results of a rapid prenatal aneuploidy screening in uncultured amniocytes using FISH are available within 24 hr. However, care has to be taken of possible pitfalls in connection with the commercially available probe sets and in the interpretation of results in general. Here we present our cut-off rates for and the way in which we handle the Aneu Vysion kit (ABBOTT/Vysis), based on the experience of 1200 studied cases. In summary, the method is reliable, and the risk for misdiagnosis is low ( approximately 0.4%). Nonetheless, the test should be used exclusively as a preamble to full chromosome analysis by microscopy.

Amniotic Fluid↗
PubMed2006

Intermingling of chromosome territories in interphase suggests role in translocations and transcription-dependent associations.

After mitosis, mammalian chromosomes partially decondense to occupy distinct territories in the cell nucleus. Current models propose that territories are separated by an interchromatin domain, rich in soluble nuclear machinery, where only rare interchromosomal interactions can occur via extended chromatin loops. In contrast, recent evidence for chromatin mobility and high frequency of chromosome translocations are consistent with significant levels of chromosome intermingling, with important consequences for genome function and stability. Here we use a novel high-resolution in situ hybridization procedure that preserves chromatin nanostructure to show that chromosome territories intermingle significantly in the nucleus of human cells. The degree of intermingling between specific chromosome pairs in human lymphocytes correlates with the frequency of chromosome translocations in the same cell type, implying that double-strand breaks formed within areas of intermingling are more likely to participate in interchromosomal rearrangements. The presence of transcription factories in regions of intermingling and the effect of transcription impairment on the interactions between chromosomes shows that transcription-dependent interchromosomal associations shape chromosome organization in mammalian cells. These findings suggest that local chromatin conformation and gene transcription influence the extent with which chromosomes interact and affect their overall properties, with direct consequences for cell-type specific genome stability.

Cells, Cultured↗
PubMed2006

Molecular cytogenetic stratification of recurrent oligodendrogliomas: utility of interphase fluorescence in situ hybridization (I-FISH).

In oligodendroglial brain tumours, losses of chromosomal material of the short arm of chromosome 1 and long arm of chromosome 19 have been shown to predict responsiveness to chemotherapy and prolonged patients' survival. Therefore, the correct diagnosis of these genetic alterations in tumours of oligodendroglial origin is particularly important. To detect deletions of 1p36 and/or 19q13.3 in oligodendroglial cells we used dual-colour I-FISH with locus-specific DNA probes. I-FISH was performed on isolated whole cell nuclei, prepared from fresh non-fixed tumour tissue samples resuspended in media and processed using a standard cytogenetic procedure, thus bypassing the problem of nuclear truncation. We examined 16 patients with histologically proved oligodendrogliomas (5x oligodendroglioma, 9x anaplastic oligodendroglioma, 2x anaplastic oligoastrocytoma). The results of molecular cytogenetic analyses were correlated with morphological and clinical findings. Molecular cytogenetic analyses were successful in 15 patients and, due to a non-adequate tissue specimen, were uninformative in one patient only. Combined deletions 1p36/19q13 were proved in 13 patients. However, in six of them additional genetic alterations typical for high-grade astrocytoma were found, which could have negative influence on the prognosis. One patient had isolated deletion of 1p36 and another had a normal genetic pattern without any chromosomal alterations. In summary, I-FISH on isolated cell nuclei is a powerful tool for detecting chromosomal aberrations in tumour cells. A systematic molecular cytogenetic analysis may advance diagnosis, prognostic stratification, and targeted treatment of patients with brain tumours.

Adult↗
PubMed1978

Occurrence of centrioles in interphasic hepatocytes of bat and chicken.

Occurrence of centrioles in non-dividing hepatocytes was examined by electron microscopy in the bat, Miniopterus schreibersi (Kuhl) and Myotis macrodactylus (Temminck), and the chicken. In both species centrioles were mostly found in the apical hyaloplasmic halo which was distinct in the chicken but rather indistinct in the bat. There was no difficulty in locating centrioles in the hepatocytes of either species. In the chicken centrioles were found in the attenuated apical cytoplasmic areas of 4-6 hepatocytes surrounding the bile canaliculus, giving us the impression that the occurrence of centrioles in non-dividing hepatocytes might be more frequent in the chicken than in the bat. The centrioles found in non-dividing bat and chicken hepatocytes apparently formed diplosomes. Neither multiple centrioles (comprising more than three) nor centriolar replications were found. Single cilium formation from the centrioles was not observed in either species.

Animals↗
PubMed1997

Effect of fixation on interphase cytogenetic analysis by direct fluorescence in situ hybridization on cell imprints.

The direct fluorescent in situ hybridization (FISH) technique using a centromerespecific DNA probe for chromosome 11 was applied on fresh tissue imprints from melanomas and pituitary adenomas. The cell imprints were fixed in acetone and formalin, and the influence of fixation was evaluated. Acetone fixed imprints gave a sharp and intense fluorescent signal in a large number of cells from both tumors without any pretreatment. In contrast, formalin fixed imprints disclosed a weak hybridization signal in a few cells even after careful enzymatic digestion. Direct FISH on acetone fixed cell imprints represents a simple and time saving technique applicable to any laboratory for the study of numerical chromosomal abnormalities.

Acetone↗