Chromatin Bodies Research Articles

Peroxisome proliferator activated receptor gamma coactivator-1 induces apoptosis in human epithelial ovarian cancer cells

To study the apoptosis-inducing effects of peroxisome proliferator activated receptor gamma (PPARgamma) coactivator-1 (PGC-1alpha) on the human epithelial ovarian cancer cell. Human epithelial ovarian cancer cell of the line HO8910 were cultured and divided into 3 groups Ad- PGC-1alpha group (infected with adenovirus. containing PGC-1alpha), Ad- GFP group (infected with adenovirus containing green fluorescent protein, and blank control group. Forty-eight hours later, light microscopy was used to observe the morphological changes of the cells, and apoptosis of the cells was observed by Hoechst staining assay. RT-PCR was used to measure the expression of intracellular Bax and Bcl-2 mRNA. Chinese hamster ovary cells of the line CHO were cultured, infected by adenovirus containing PGC-1alpha and GFP and negative adenovirus respectively, and then underwent Hoechst staining assay and flow cytometry. Light microscopy and Hoechst staining assay showed that there were many morphological characteristics of apoptosis including compaction and margination of nuclear chromatin, nuclear fragments, and apoptotic bodies in the Ad- PGC-1alpha. RT-PCR showed the mRNA expression of intracellular Bax was up-regulated by 1.5 times and the mRNA expression of Bcl-2 was down-regulated by 69% in the Ad- PGC-1alpha group. However, PGC-1alpha treatment did not induce apoptosis in the CHO cells. PGC-1alpha induces apoptosis in epithelial ovarian cancer cells in vitro, which may be a result of up-regulation of the pro-apoptotic gene Bax and down-regulation of the anti-apoptotic gene Bcl-2. And over-expression of PGC-1alpha results in no apoptosis in CHO.

Changes in chromosome positioning may contribute to the development of diseases related to X‐chromosome aneuploidy

The radial positions of the centromeric regions of chromosomes 1 and X were determined in normal male fibroblasts (XY) and in fibroblasts from a patient with a rare case of XXXXY polysomy. The centromeric regions and presumably the whole territories of active X chromosomes were demonstrated to occupy similar, although not identical, positions in XY and XXXXY cells. The centromeres of inactive X chromosomes (Barr bodies) were located closer to the nuclear periphery as compared with the centromeres of active X chromosomes. In addition, it was established that the nuclear radial position of gene-rich chromosome 1 was changed in XXXXY cells as compared to normal XY cells. The data are discussed in the context of the hypothesis postulating that changes in nuclear positioning of chromosomal territories induced by the presence of extra copies of individual chromosomes may contribute to the development of diseases related to different polysomies.

Fibrillogenic amylin evokes the apoptosis of human mesangial cells

Objective To explore the apoptotic role of amylin on human mesangial cell (MC). Materials and methods Primarily cultured human MCs were applied and treated with fresh amylin preparation. Human MCs were identified by the morphology and immunofluorescence staining. The apoptotic cells were determined by ultrastructure changes, TUNEL, and DNA fragmentation analysis. Propidium iodide staining and flow cytometry was employed for quantitative measurement of apoptosis. Results Under the light and transmission electronic microscopy (TEM), the human MCs with condensed chromatin, plasma shrinkage, marginated nuclear chromatin or apoptotic body were observed in amylin-treated MCs. Positive TUNEL staining, hypolipoid DNA peak, and typical DNA “ladder” pattern were also detected in amylin-treated MCs. Quantitative analysis of the apoptotic MCs showed that human amylin induced an increase of the percentage of apoptotic cells in a dose-dependent manner. Amylin nano-scale fibrils (5–18 nm) in diameter were detected in the cultured solution using negative staining under the TEM. Compared to the control, no significant changes of lactate dehydrogenase release were observed in amylin-treated MCs ( P > 0.05). Conclusions Fibrillogenic amylin evokes the apoptosis of human MCs in vitro, which may explain the mechanism of the hypocellular mesangial damage and progressive glomerulosclerosis of the patients with diabetic nephropathy.

Oncosis, the possible cell death pathway in astrocytes after focal cerebral ischemia

Swelling of astrocytes at early stage of cerebral ischemia has been reported, however, the fate and the cell death pathway of astrocytes are still unclear. Focal cerebral ischemia was induced in Sprague–Dawley rats by permanent occlusion of middle cerebral artery for 3 to 48 h. Haematoxylin and eosin (HE) staining, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), glial fibrillary acidic protein (GFAP), caspase-3 immunostaining, and double-staining with TUNEL and GFAP were carried out on consecutive sections. The ultrastructure was revealed by electron microscopy. Using electron microscope, apoptotic neurons were confirmed with condensed chromatin and apoptotic bodies. In the core of the infarct, clumps of heterochromatin around the edge of nucleus, vacuolar degeneration of the nucleus and leakage of chromatin were demonstrated at 3, 6, and 12 h respectively in the swelling astrocytes, which accorded with the process of oncosis; in the peripheral zone of the infarct, reactive astrocytes with nuclear membranes preserved demonstrated increased cell size and number and coexisted with oncotic astrocytes. Scattered GFAP-positive cells and ubiquitous caspase-3-positive cells were found in the core after 12 h following cerebral ischemia, and no cells positive for double-staining with TUNEL and GFAP were found in the ischemic regions, indicating that most GFAP-positive astrocytes did not die by apoptosis. Findings from present study demonstrate that after cerebral ischemia, oncosis may be the possible cell death pathway of astrocytes in the ischemic region, and oncotic astrocytes coexist with reactive astrocytes in the peripheral zone.

Sudden death of a young woman due to aortic dissection caused by Turner's syndrome

A 24-year-old woman was found dead in her bed. There had been an episode of fainting with cervicodynia 1 day before death but no significant past medical history, except for menstrual irregularities. Post-mortem examination revealed that death was due to hemopericardium caused by rupture of the ascending aorta by thoracic aortic dissection (Stanford type A). Microscopically, weakness of the aorta was due to cystic medial necrosis. On external examination, short stature, a short neck and multiple pigmented nevi were observed, while internal examination revealed coarctation of the aorta and funicular ovaries. Examination of the X chromatin showed a decrease in numbers of Barr bodies in the tissues, and a 45,X/46,XX mosaicism was suspected. It is concluded that the cause of death was aortic dissection due to Turner's syndrome.

Fine structure of spermiogenesis, spermatozoa and spermatophore of Saxidromus delamarei (Saxidromidae, Actinotrichida, Acari)

Ultrastructural details of spermiogenesis, spermatozoa and the spermatophore of the early derived actinedid mite Saxidromus delamarei are described. Spermatids and mature sperm cells are provided with up to four acrosomal complexes and nuclei derivatives (chromatin bodies). Due to this reason, the sperm cells may be classified as synspermia, a sperm type found only in some spiders until now. The acrosomal complex is composed of a remarkably complicated vacuole and filament. Other peculiarities of sperm structure correspond to those found in prostigmatic mites, i.e. penetration of the chromatin body by the acrosomal filament and the presence of peripheral invaginations of the plasmalemma. The sperm cells are covered by a thin secretion layer of probably proteinaceous material. Stalked spermatophores are rather large, but simply structured and contain relatively few sperm cells. The results are discussed taking systematical and behavioural aspects into account. In particular, it is suggested that the peculiar mating behaviour of these mites secures both sperm transfer and first male's sperm priority and that this allowed reduction of sperm numbers.

Global analysis of chromosome X gene expression in primary cultures of normal ovarian surface epithelial cells and epithelial ovarian cancer cell lines

The interpretation of loss of heterozygosity (LOH) in cancers is complicated as genes that map to LOH regions may be transcriptionally active (Xa) or inactive (Xi) due to X chromosome inactivation (XCI). We have analyzed the chromosome X transcriptome in four epithelial ovarian cancer (EOC) cell lines (TOV21G, TOV81D, TOV112D, and OV90) and 12 primary cultures of normal ovarian surface epithelial (NOSE) cells in relation to chromosome X integrity. Two-way comparative analysis using HuGeneFL Affymetrix GeneChips of TOV21G, TOV81D and OV90 relative to the NOSE samples was highly correlated (> 89%) in contrast to that of TOV112D (56-69%). TOV112D, followed by TOV21G, exhibited the largest number of up-regulated genes. XIST expression by RT-PCR was not detectable in TOV112D or TOV21G. Allele-specific transcription by cDNA sequence analysis of genes known to be subjected to XCI revealed maintenance of XCI in TOV81D and OV90, but not TOV21G. Biallelic expression could not be assessed in TOV112D due to reduction to hemizygosity of chromosome X. Chromosome X rearrangements were observed in FISH analysis of TOV112D and TOV21G, and both of these EOC cell lines were negative for Barr body analysis. The differentially expressed genes did not appear to map to any particular region of the X chromosome in any EOC cell line. The absence of XIST expression is consistent with Barr body loss in TOV112D and TOV21G. The combined evidence is consistent with two proposed mechanisms to account for absence of Xi in female cancers: Xi loss followed by Xa duplication (exemplified by TOV112D) and transcriptional reactivation of Xi (exemplified by TOV21G). Despite an alteration in XIST expression and differences in allelic content in the EOC cell lines, the chromosome X transcriptome was modified modestly when compared with that of NOSE samples.

Supranucleosomal Organization of Chromatin Fibers in Nuclei ofDrosophilaS2 Cells

Earlier, the interphase chromatin structures could not be visualized due to the stickiness of the nuclear material. We have reduced stickiness by the reversal of permeabilization allowing the isolation and microscopic imaging of interphase chromatin structures. By using a high resolution of synchronization, collecting 36 elutriation fractions, we show that major intermediates of chromatin condensation include: (a) decondensed veillike chromatin at the unset of the S phase (2.0-2.2 C-value), (b) polarization of veiled chromatin (2.2-2.6 C), (c) fibrous chromatin (2.6-3.0 C), chromatin bodies (3.0-3.3 C), early precondensed chromosomes (3.3-3.6). The compaction of Drosophila chromosomes did not reach that of the mammalian cells in the final stage of condensation (3.6-4.0 C). Drosophila chromosomes consist of smaller units called rodlets. Results demonstrate that nucleosomal chromatin ("beads on string") does not form a solenoid structure; rather, the topological arrangement consists of meandering and plectonemic loops.

Condensed Chromatin, Cell Thermoregulation and Human Body Heat Conductivity

Earlier we put out a proposal on possible participation of condensed chromatin (CC) in cell thermoregulation; CC being the densest domain in a cell, apparently conducts heat between the cytoplasm and nucleus when there is difference in temperature between them. This hypothesis can be checked at the level of cells or organisms. Experimentally we have managed to establish that at the level of organisms there is a broad intra population variability of human body heat conductivity (BHC). It is shown that these individual differences in the BHC are attributed to the amount of chromosomal Q-heterochromatin regions (Q- HRs) in their genome. It is assumed that, possibly, the biological role of the Q-HRs in the interphase nucleus of the cell is in intensification of the CC compacting thus increasing its heat conductivity (HC). On the HC of CC, correspondently on the amount of Q-HRs in the genome, depends the speed of leveling the difference of temperature between the cytoplasm and nucleus, i.e. the cell thermoregulation. From the HC of the cells the HC of the whole body is made up. This is a physical condition, where the physiological thermoregulation is realized, which is assigned for keeping relative temperature constancy in the inner medium of the organism by leveling the temperature difference in different parts of the body.

Successful Reversion of Refractory Paroxysmal Nocturnal Hemoglobinuria (PNH) by Allogeneic Peripheral Blood Stem Cell Transplantation.

Objective: To observe the safety and effect of allogeneic peripheral blood stem cell transplantation (allo-PBSCT) on hematopoietic reconstitution of refractory paroxysmal nocturnal hemoglobinuria(PNH)and reversion of hemolysis.Methods: A 20 years old female patient diagnosed with PNH complicated with cerebral infarction underwent allo-PBSCT. The donor was the patient's 18 years old biological brother. Six loci on HLA-A, B, and DRB1 were completely matched with those of the recipient. Four days after the administration of G-CSF (250 μg/day) to the recipient, peripheral blood stem cells (PBSCs) were collected from the donor for 2 consecutive days. A total volume of 112 ml was harvested. A median nucleated cell (MNC) count of 6.2×108/kg with 2.4×106 /kg of CD34+ cells were actually administered to the recipient. The standard conditioning regimen was busulfan/cyclophosphamide (BU/CY). The recipient was started on intravenous infusion (IV) of cyclosporine A (CSA) on Day -1 and a plasma concentration of 150–250 ng/L was maintained. IV methotrexate (MTX) 10mg/m2 was given on Days +1, +3, +6, and +11. Oral mycophenolate mofetil(MMF)was administrated from Days +1 to +28 for prophylaxis of acute graft-versus-host disease (GVHD). If the concentration of hemoglobin (Hb) decreased below 70 g/L and/or platelet count (BPC) below 15×109 /L, the recipient was transfused with 60Co 25cGy irradiated and leuko-depleted red blood cells (RBCs) and/or single-donor platelets. Starting from Day +3, G-CSF was given to the recipient until white blood cells (WBC) increased to above 3.0× 109 /L. EPO and Interleukin-11 were also administrated to stimulate the proliferation of the progenitors of erythrocytes and megakaryocytes.Results: DNA-STR on Day +33 indicated a complete chimerism in the recipient. Chromatin bodies were observed as 46XY on Day +45. CD55/CD59 expression on the membranes of leucocytes and erythrocytes, hemolysis index, and coagulation parameters returned to normal. The patient has been relapse-free for one year since the time of discharge.Conclusion: 1. When conventional therapy with hormone and immunosuppressant becomes unsuccessful to control PNH, allo-PBSCT may be an alternative measure with promising effect. 2. In comparison to the reduced dose regimen, the standard dose regimen of BU/CY shown lower rate of relapse. 3. To decrease the mortality rate related to transplantation, allo-PBSCT needs to be considered as early as possible for refractory paroxysmal nocturnal hemoglobinuria.

Probing the W chromosome of the codling moth, Cydia pomonella, with sequences from microdissected sex chromatin

The W chromosome of the codling moth, Cydia pomonella, like that of most Lepidoptera species, is heterochromatic and forms a female-specific sex chromatin body in somatic cells. We collected chromatin samples by laser microdissection from euchromatin and W-chromatin bodies. DNA from the samples was amplified by degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) and used to prepare painting probes and start an analysis of the W-chromosome sequence composition. With fluorescence in situ hybridization (FISH), the euchromatin probe labelled all chromosomes, whereas the W-chromatin DNA proved to be a highly specific W-chromosome painting probe. For sequence analysis, DOP-PCR-generated DNA fragments were cloned, sequenced, and tested by Southern hybridization. We recovered single-copy and low-copy W-specific sequences, a sequence that was located only in the W and the Z chromosome, multi-copy sequences that were enriched in the W chromosome but occurred also elsewhere, and ubiquitous multi-copy sequences. Three of the multi-copy sequences were recognized as derived from hitherto unknown retrotransposons. The results show that our approach is feasible and that the W-chromosome composition of C. pomonella is not principally different from that of Bombyx mori or from that of Y chromosomes of several species with an XY sex-determining mechanism. The W chromosome has attracted repetitive sequences during evolution but also contains unique sequences.

Linear Connection of Condensing Chromosomes in Nuclei of Synchronized CHO Cells

Reversibly permeabilized cells allowed the analysis of intermediates of large-scale chomatin condensation in a cell cycle-dependent manner. This paper summarizes major intermediates of chromatin condensation and visualizes connectivity between different forms of chromosomes. At the early S phase the veil-like fibrillary chromatin is supercoiled to form chromatin bodies representing the earliest visible chromosomes. Supercoiling results in a chromatin fiber, which turns to rope (thick fiber) forming loops and chromatin ribbon. The elongated shape of prechromosomes indicates that they are arranged head to tail. Bent forms (loop, c-, and v-shaped structures) of interphase chromosomes open at the end of S phase. Linear arrangement of chromosomes was observed to the end of the condensation process, suggesting that connectivity of chromosomes is maintained throughout the cell cycle.

Cellular responses of the ciliate, Tetrahymena thermophila, to far infrared irradiation

Infrared rays from sunlight permeate the earth's atmosphere, yet little is known about their interactions with living organisms. To learn whether they affect cell structure and function, we tested the ciliated protozoan, Tetrahymena thermophila. These unicellular eukaryotes aggregate in swarms near the surface of freshwater habitats, where direct and diffuse solar radiation impinge upon the water-air interface. We report that populations irradiated in laboratory cultures grew and mated normally, but major changes occurred in cell physiology during the stationary phase. Early on, there were significant reductions in chromatin body size and the antibody reactivity of methyl groups on lysine residues 4 and 9 in histone H3. Later, when cells began to starve, messenger RNAs for key proteins related to chromatin structure, intermediary metabolism and cellular motility increased from two- to nearly nine-fold. Metabolic activity, swimming speed and linearity of motion also increased, and spindle shaped cells with a caudal cilium appeared. Our findings suggest that infrared radiation enhances differentiation towards a dispersal cell-like phenotype in saturated populations of Tetrahymena thermophila.

‘Your true and proper gender’: the Barr body as a good enough science of sex

In the late 1940s, a microanatomist from London Ontario, Murray Barr, discovered a mark of sex chromosome status in bodily tissues, what came to be known as the ‘Barr body’. This discovery offered an important diagnostic technology to the burgeoning clinical science community engaged with the medical interpretation and management of sexual anomalies. It seemed to offer a way to identify the true, underlying sex in those whose bodies or lives were sexually anomalous (intersexuals, homosexuals and transsexuals). The hypothesis that allowed the Barr body to stand in for ‘chromosomal’ or ‘genetic’ sex was provisional, but it supported the expectation that genetic information established one’s primary identity, and the conviction that the animal world could be neatly divided into two, and only two, sexes. Ultimately, this provisional hypothesis, and its status as an unambiguous arbiter of true sex, was overturned. But during much of the 1950s, Barr’s thesis about the identity of the Barr body was consistent with a coherent set of theories and evidence explaining sexual development and sexual pathology. Though provisional, the scientific status of the sex chromatin within this system of knowledge was good enough to support a flourishing research enterprise in the clinical sciences.

The X chromosome is organized into a gene-rich outer rim and an internal core containing silenced nongenic sequences.

We investigated whether genes escape X chromosome inactivation by positioning outside of the territory defined by XIST RNA. Results reveal an unanticipated higher order organization of genes and noncoding sequences. All 15 X-linked genes, regardless of activity, position on the border of the XIST RNA territory, which resides outside of the DAPI-dense Barr body. Although more strictly delineated on the inactive X chromosome (Xi), all genes localized predominantly to the outer rim of the Xi and active X chromosome. This outer rim is decorated only by X chromosome DNA paints and is excluded from both the XIST RNA and dense DAPI staining. The only DNA found well within the Barr body and XIST RNA territory was centromeric and Cot-1 DNA; hence, the core of the X chromosome essentially excludes genes and is composed primarily of noncoding repeat-rich DNA. Moreover, we show that this core of repetitive sequences is expressed throughout the nucleus yet is silenced throughout Xi, providing direct evidence for chromosome-wide regulation of "junk" DNA transcription. Collective results suggest that the Barr body, long presumed to be the physical manifestation of silenced genes, is in fact composed of a core of silenced noncoding DNA. Instead of acting at a local gene level, XIST RNA appears to interact with and silence core architectural elements to effectively condense and shut down the Xi.

Common Pathway of Chromosome Condensation in Mammalian Cells

Chromatin folding in the interphase nucleus is not known. We compared the pattern of chromatin condensation in Indian muntjac, Chinese hamster ovary, murine pre B, and K562 human erythroleukemia cells during the cell cycle. Fluorescent microscopy showed that chromosome condensation follows a general pathway. Synchronized cells were reversibly permeabilized and used to isolate interphase chromatin structures. Based on their structures two major categories of intermediates were distinguished: (1) decondensed chromatin and (2) condensed chromosomal forms. (1) Chromatin forms were found between the G1 and mid-S phase involving veil-like, supercoiled, fibrous, ribboned structures; (2) condensing chromosomal forms appeared in the late-S, G2, and M phase, including strings, chromatin bodies, elongated pre-chromosomes, pre-condensed chromosomes, and metaphase chromosomes. Results demonstrate that interphase chromosomes are clustered in domains; condensing interphase chromosomes are linearly arranged. Our results raise questions related to telomer sequences and to the chemical nature of chromosome connectivity.

An ultrastructural investigation of the testes and spermiogenesis in Myobia murismusculi (Schrank) (Acari, Actinedida: Myobiidae)

Summary The stages of spermiogenesis in Myobia murismusculi were investigated on the basis of ultrastructural analysis of both the testes and the female organs: receptaculum seminis and seminal duct. The walls of the testes consist of a thin epithelial layer. Germ and secretory cells lie free in the lumen of the testes. In the early stages of differentiation, both cell types represent clusters of sister cells joined by intercellular bridges. Each secretory cell contains prominent RER and Golgi complex, which produce single dense granule. Growing gradually the granule fills the whole volume of the cell's cytoplasm. Mature secretory cells disintegrate and the secretory product discharges into the testicular lumen. The germ cells are represented by the early, the intermediate and the late spermatids as well as the immature sperm (prospermia). Neither spermatogonia nor meiotic figures were observed in adult males. As spermiogenesis starts, numerous narrow invaginations of the outer membrane (peripheral channels) develop on the cell surface. They form a wide circumferential network connected to pinocytotic vesicles. Owing to the secretory activity of the Golgi complex, a large acrosomal granule is formed in the early spermatids. A long acrosomal filament runs along the intranuclear canal. Nuclear material condenses and forms two spherical bodies of different electron density. The lighter one can be observed until the stage of the late spermatids, when the nuclear envelope almost completely disappears. The electron-dense nuclear body transforms into a definite chromatin body, which is observed in the mature sperm as a cup-shaped structure. The late spermatids are characterized by the presence of a large electronlucent vacuole, which seems to be unique for the process of spermiogenesis in Actinedida. After the spermia enter the female genital tract, the peripheral channels disappear as well as the vacuole. The cells form long amoeboid arms with a special microtubular layer underneath the plasma membrane. The chromatin body is encircled by a large acrosomal granule of complex shape provided by long extensions running deep into the cytoplasm. The cytoplasm contains no organelles except for a group of unmodified mitochondria in the post-nuclear region. The main characteristics of the Myobia spermiogenesis are discussed with regard to other actinedid mites.

Evaluation of breast tumour sex chromatin (Barr body) as an index of survival and response to pituitary ablation.

Abstract In a retrospective study of 36 patients with advanced oreast cancer relationships were found between the cumour Barr body count and the response to pituitary ablation and the length of survival. High tumour Barr body counts were associated with long survival after treatment, and low counts with short survival. (P < 0.001). High counts were also associated with a good response to pituitary ablation, and low counts with a poor response (P < 0.01). The tumour Barr body count gave possibly better prediction than urinary steroid measurement.

Double minutes, cytogenetic equivalents of gene amplification, in human neoplasia—a review

Double minutes are tiny spherical chromatin bodies of a few mega-base pairs of size which are found occasionally in hematopoietic neoplasia and more or less often in human solid tumors. They have been associated with worse prognosis and poor outcome of the malignancies where present. With the beginning era of molecular cytogenetics they could be defined as cytogenetic equivalents of amplified DNA sequences. The identification of involved chromosomal segments and their molecular nature led to the development of molecular genetic techniques for a rapid and reliable detection of prognostically important oncogene amplifications in human tumors and,as a consequence, to gene-targeted therapy.

Whole-genome chromosome distribution during nuclear fragmentation of giant trophoblast cells of Microtus rossiaemeridionalis studied with the use of gonosomal chromatin arrangement

Gonosomal chromatin bodies (GCBs), i.e. blocks of condensed chromatin consisting of heterochromatized region of the sex chromosomes of the field vole M. rossiaemeridionalis, were used as a natural interphase chromosome marker in order to clarify the regularities of GCB rearrangement during nuclear fragmentation of secondary giant trophoblast cells (SGTCs) at the end of their differentiation. Cytophotometrical measurements of DNA content in the nuclei, nuclear fragments and simultaneously in the GCBs were made in the secondary giant SGTCs of field vole M. rossiaemeridionalis. In most cases 1 to 2 GCBs get into the nuclear fragments at different ploidy levels. In the nuclear fragments, GCB DNA content decreased mostly proportionally to DNA content in the whole fragments corresponding to 2c, 4c and 8c. The data obtained demonstrate a regular whole-genome chromosome distribution into nuclear fragments. A possible mechanism of nuclear fragmentation that largely ensures a balanced genome in nuclear fragments is discussed.