The condensin complex is essential for amitotic segregation of bulk chromosomes, but not nucleoli, in the ciliate Tetrahymena thermophila

The macronucleus of the binucleate ciliate Tetrahymena thermophila contains fragmented and amplified chromosomes that do not have centromeres, eliminating the possibility of mitotic nuclear division. Instead, the macronucleus divides by amitosis with random segregation of these chromosomes without detectable chromatin condensation. This amitotic division provides a special opportunity for studying the roles of mitotic proteins in segregating acentric chromatin. The Smc4 protein is a core component of the condensin complex that plays a role in chromatin condensation and has also been associated with nucleolar segregation, DNA repair, and maintenance of the chromatin scaffold. Mutants of Tetrahymena SMC4 have remarkable characteristics during amitosis. They do not form microtubules inside the macronucleus as normal cells do, and there is little or no bulk DNA segregation during cell division. Nevertheless, segregation of nucleoli to daughter cells still occurs, indicating the independence of this process and bulk DNA segregation in ciliate amitosis.     

Light and electron microscopic radioautographic studies on macromolecular synthesis in amitotic hepatocytes of aging mice.

The problem on cell divisions whether cells proliferate by mitosis or amitosis has been the heated controversy among many biologists since the late 19th century. We confirmed by extensive experiments since the mid 20th century that all the cells proliferated by mitosis not by amitosis but that amitosis actually existed in some glandular cells such as hepatocytes or pancreatic acinar cells which showed only amitotic nuclear divisions without cytoplasmic division producing binucleate cells in several kinds of experimental animals. We further verified that such amitotic cells did not synthesize macromolecular compounds incorporating macromolecular precursors such as 3H-thymidine for DNA, 3H-uridine for RNA or 3H-leucine for proteins. Recent experiments at the end of 20th century using many groups of aging mice, from fetal day 19 to postnatal month 24, injected with such precursors, amitotic cells and resulting binucleate cells in the hepatocytes were detected by electron microscopic radioautography and compared to mononucleate cells. The results demonstrated that only a few hepatocytes showing amitotic nuclear division were found labelled with the 3 precursors demonstrating DNA, RNA and protein synthesis. However, the numbers of silver grains showing incorporations of labelled precursors in respective amitotic cells were very few. It was clarified that the amitotic cells did not synthesize such macromolecules as mononucleate hepatocytes did. On the other hand, more binucleate cells were found than the amitotic cells. DNA synthesis of mononucleate and binucleate hepatocyte nuclei was observed at perinatal stage and disappeared at adult stage. The labeling index of mononucleate hepatocytes was greater than that of binucleate hepatocytes. RNA and protein syntheses in karyoplasm and cytoplasm in both mononucleate and binucleate cells increased from perinatal stage, reaching the maxima at adult stage then decreased to senescent stage. Grain counts revealed that synthesized RNA and proteins were more in binucleate cells than mononucleate cells at respective aging stages.     

Chromosome Copy Number Variation and Control in the Ciliate Chilodonella uncinata

Copy number variations are widespread in eukaryotes. The unusual genome architecture of ciliates, in particular, with its process of amitosis in macronuclear division, provides a valuable model in which to study copy number variation. The current model of amitosis envisions stochastic distribution of macronuclear chromosomes during asexual reproduction. This suggests that amitosis is likely to result in high levels of copy number variation in ciliates, as dividing daughter cells can have variable copy numbers of chromosomes if chromosomal distribution during amitosis is a stochastic process. We examined chromosomal distribution during amitosis in Chilodonella uncinata, a ciliate with gene-size macronuclear chromosomes. We quantified 4 chromosomes in evolving populations of C. uncinata and modeled the amitotic distribution process. We found that macronuclear chromosomes differ in copy number from one another but that copy number does not change as expected under a stochastic process. The chromosome carrying SSU increased in copy number, which is consistent with selection to increase abundance; however, two other studied chromosomes displayed much lower than expected among-line variance. Our models suggest that balancing selection is sufficient to explain the observed maintenance of chromosome copy during asexual reproduction.     

A probabilistic model of mosaicism based on the histological analysis of chimaeric rat liver

The analysis of pattern development in mosaic and chimaeric animals has provided insight into a number of developmental problems. In order to aid the understanding of the dynamics of the development of mosaic tissues, a computer simulation of the generation of a mosaic tissue was created using simple probabilistic decisions. Results of quantitative analysis of the simulated mosaicism were compared with chimaeric liver. Chimaeric animals were produced by morula aggregation between histologically distinguishable strains of congenic rats. The livers of these animals revealed a pattern of patchy mosaicism unrelated to either acinar or lobular architecture of the organ. Independent quantifiable parameters were correlated and compared between the simulation and chimaeric liver tissue. This analysis showed that extensive cell migration is not required to develop finely variegated mosaic tissue and that the patterns of mosaicism observed could have resulted from tissue development in which as few as three reiterated decisions were required. First, the simulation established anlagen of two cell types of various specified proportions with randomly chosen placement. Second, in each generation of the simulation the order in which the cells divided was established randomly. Third, there was a random choice of the direction of placement of the daughter cell. The quantitative relationships between the proportion of cell types, the area of patches and the number of patches per unit area was consistent between the simulation and the chimaeric tissue.     

Suspension Culture of Endosperm Callus Cell and Amitosis In Vitro

The endosperm calli were induced on MS basic medium supplemented with lppm 2,4-D, 0.5ppm KT and 5% sucrose. The medium which contained lppm BAP, 0.1ppm NAA and 2% sucrose was used for cell suspension culture. In suspension cell culture, amitosis of cleavage division of nucleus have been observed after 5 days of culture. First the nuclear membrane and nucleolus disappeared. The crevice appeared in the center of the nucleus, and the nucleus divided into two daughter nuclei of similar size and each with a nucleolus. The daughter nucleus resembled an eye in shape. Following the emergence of cell wall, the two new unequal cells were produced. Such amitotic division proceeded repeatedly until the callus developed and eventually plantlet regenerated.     

小麦离体花药中花粉核无丝分裂的电子显微镜观察

用电子显微镜观察到,在离体培养的小麦花药中,花粉细胞在脱分化分裂时,除了进行有丝分裂之外,还存在着两种类型的无丝分裂——劈裂(cleavage)和碎裂(fragmentation)。它们都是通过核膜的内陷实现的。认为劈裂式无丝分裂导致游离核花粉的形成,碎裂式无丝分裂导致微核花粉的形成,两者都可引起亚倍体和非整倍体植株的产生。     

An Ultrastructural Study on Triggering of Cell Division in Young Pollen Protoplast Culture of Hemerocallis fulva L.

The ultrastructural changes of young pollen protoplasts under culture condition in Hemerocallis fulva were studied. In comparison with the original pollen grains, the pollen protoplasts had been completely deprived of pollen wall, but kept the internal structure intact, including a large vacuole, a thin layer of cytoplasm and a peripherally located nucleus. After 8 days of culture a few pollen protoplasts were triggered to cell division: some of them were just undergoing mitosis with clearly visible chromosomes and spindle fibers; the others already divided into 2-celled units. The two daughter cells were equal or unequal in size but with similar distribution of organelles inside. Besides cell division, there were also free nuclear division, amitosis and formation of micronuclei indicating a diversity of division modes in pollen protoplast culture, A series of changes occurred during the process of induction of cell division, such as locomotion of the nucleus toward the central position, disappearence of the large vacuole, increase of electron density of cytoplasm, increase and activation of organelles, diminishing of starch granules in plastids, etc. However, the regeneration of surface wall was not sufficient it contained mostly vesicles with only a few microfibrits. The wall separating the two daughter cells were either complete or incomplete. The weak capability of wall formation is supposed to be one of the major obstacles which has so far restricted sustained cell divisions of young pollen protoplasts under current culture condition.     

Dissection of the insulin-sensitizing effect of liver X receptor ligands

The liver X receptors (LXRalpha and beta) are nuclear receptors that coordinate carbohydrate and lipid metabolism. Treatment of insulin-resistant mice with synthetic LXR ligands enhances glucose tolerance, inducing changes in gene expression expected to decrease hepatic gluconeogenesis (via indirect suppression of gluconeogenic enzymes) and increase peripheral glucose disposal (via direct up-regulation of glut4 in fat). To evaluate the relative contribution of each of these effects on whole-body insulin sensitivity, we performed hyperinsulinemic-euglycemic clamps in high-fat-fed insulin-resistant rats treated with an LXR agonist or a peroxisome proliferator-activated receptor gamma ligand. Both groups showed significant improvement in insulin action. Interestingly, rats treated with LXR ligand had lower body weight and smaller fat cells than controls. Insulin-stimulated suppression of the rate of glucose appearance (Ra) was pronounced in LXR-treated rats, but treatment failed to enhance peripheral glucose uptake (R'g), despite increased expression of glut4 in epididymal fat. To ascertain whether LXR ligands suppress hepatic gluconeogenesis directly, mice lacking LXRalpha (the primary isotype in liver) were treated with LXR ligand, and gluconeogenic gene expression was assessed. LXR activation decreased expression of gluconeogenic genes in wild-type and LXRbeta null mice, but failed to do so in animals lacking LXRalpha. Our observations indicate that despite inducing suggestive gene expression changes in adipose tissue in this model of diet-induced insulin resistance, the antidiabetic effect of LXR ligands is primarily due to effects in the liver that appear to require LXRalpha. These findings have important implications for clinical development of LXR agonists as insulin sensitizers.     

Cytoplasmic manifestation of the nuclear membrane's hormone binding capacity during cell division

Binding of insulin and thyrotropic hormone (TSH) to the nuclear membrane of Chang liver cells was demonstrated by qualitative and quantitative cytofluorimetry, which failed to substantiate a similar binding affinity for BSA. It appears that in the dividing cell the binding structures (receptors) of the nuclear membrane migrate in the cytoplasm together with the chromosomes by the end of the prophase and become reorganized in the nucleus around the telophase. The fluorescence which indicated binding also appeared in the midbody region during division of the two daughter cells. These experimental observations strongly suggest that, after cell division, only part of the nuclear membrane's receptor complement has to be resynthesized in the daughter cells, because the receptor number required by a single cell is conserved in cytoplasmic membrane details of nuclear membrane origin.     

Cytoplasmic manifestation of the nuclear membrane's hormone binding capacity during cell division

Summary Binding of insulin and thyrotropic hormone (TSH) to the nuclear membrane of Chang liver cells was demonstrated by qualitative and quantitative cytofluorimetry, which failed to substantiate a similar binding affinity for BSA. It appears that in the dividing cell the binding structures (receptors) of the nuclear membrane migrate in the cytoplasm together with the chromosomes by the end of the prophase and become reorganized in the nucleus around the telophase. The fluorescence which indicated binding also appeared in the midbody region during division of the two daughter cells. These experimental observations strongly suggest that, after cell division, only part of the nuclear membrane's receptor complement has to be resynthesized in the daughter cells, because the receptor number required by a single cell is conserved in cytoplasmic membrane details of nuclear membrane origin.     

Observations on induced amitosis in Acanthamoeba

Methods are described for the induction of amitotic cell division in Acanthamoeba rhysodes. Induced amitotic cell division in this organism is similar to normal cytokinesis in many respects, however, the nucleus is partitioned during its interphase state so that the daughter products of amitosis are not viable. It is proposed that the induction of amitotic cell division causes the amoeba to produce the normal cytoplasmic components responsible for cell division in the absence of nuclear mitosis. This is not a normal stage in the amoeba's life cycle and it appears to be a genetic defect unique to this strain of Acanthamoeba.Evidence is presented that induction of amitotic cell division requires protein synthesis but not ribonucleic acid synthesis. Further, induced amoebae require a period of adhesion to a foreign substrate before they are capable of amitosis. The pattern of amitotic cell division could be interpreted as a segregation of discrete cytoplasmic units, generated during induction.     

A modified immunohistochemical procedure for the detection of estrogen receptor in mouse tissues

A horseradish peroxidase (HRP) labeled antibody method was developed for use with a monoclonal antibody to detect estrogen receptor (ER) in mouse tissue. Combined use of HRP labeled F(ab')2 fragment absorbed with mouse liver protein to minimize background staining and imidazol-DAB reaction gave the most reliable and sensitive immunostaining. The method was applied to uterine, vaginal, pituitary and liver tissues in ovariectomized adult mice. In uterus and vagina, ER was recognized in nuclei of epithelial cells, stromal cells and smooth muscle cells of the muscle layer and blood vessels. Liver tissue showed positive nuclear immunostaining in parenchymal cells; however, no reaction was present in endothelial cells, Kupffer cells, bile ductal cells, and smooth muscle cells of blood vessels. ER was localized in the nuclei of anterior pituitary cells while weak reaction was also recognized in cells of the intermediate lobe. No staining was detected in the posterior pituitary. Results demonstrate that both occupied and unoccupied ER are localized in the cell nucleus from several target tissues. Weak immunostaining in samples could not be enhanced by multiple procedures. It is suggested that nuclear ER is partially hidden by nuclear components such as nucleic acid and chromatin proteins.     

The Nucleolus in the Induced Amitosis

Nucleolar cycles were related to mitotic and amitotic phasesin meristematic cells of Allium cepa L. roots growing in controlconditions and in others where amitosis was induced by a 6-htreatment with ethidium bromide. The induced amitosis in these cells was followed by an unbalanceddistribution of chromosomal material between the two independentdaughter cells resulting from it. The first discrepancy between mitosis and amitosis was foundat the level of prophase nucleolar disorganization, which wasgreatly modified. The nucleolar cycle was permanently stoppedin amitosis even when recovery from treatment was allowed. Thekinetics of amitosis appearance in the meristem suggest a decisionpoint located round about mid-prophase. This decision pointseems to involve nucleolar function in prophase. These observationsconfirm the fact that prophase is one of the decisive stagesin the differentiation of a cell.     

A cytological study of Nematospora coryli pegl

A study is made of nuclear division in Nematospora coryli, a pathogenic yeast. The DNA of cells (grown on a V-8-medium) was stained with leuco-basic fuchsin (Feulgen test) at pH 3.5. After budding has started the rounded nucleus elongates and some differentiation into chromosomes is perceptible. A few slides suggest the number of chromosomes being 4. After some time the nucleus appears to have duplicated. This nucleus migrates towards the isthmus between mother cell and bud. In the isthmus, or just in front of it, the two daughter nuclei proceed to disjoin and move along each other to opposite directions. One daughter nucleus moves into the bud; the other one migrates back into the mother cell.Samples from synchronously growing cultures show that a fraction of the young yeast cells are destined to grow out to asci, in which after about 6 hours the presence of bivalents seems highly probable. The succeeding nuclear divisions take place in the same way as described for the vegetative cells and stop when the majority of the enlarged asci contain 8 nuclei.Problems of haploidy and diploidy are discussed.Small, densely stained bodies are observed in certain vegetative and some meiotic stages. As these bodies contain DNA, their function and possible homology with centrioles is discussed.     

A method for determining ploidy distributions in liver tissue by stereological analysis of nuclear size calibrated by flow cytometric DNA analysis

A method is presented for determining ploidy distributions in mouse liver from image analysis with stereological estimations of nuclear size in tissue sections. Nuclear profile distributions obtained from profile measurements were subjected to a mathematical unfolding procedure in order to obtain the nuclear size distributions. Based on the assumption that nuclear size increases monotonically with nuclear DNA content, flow cytometric DNA analysis of suspensions of liver cell nuclei was used to calibrate the method, thus yielding the mean nuclear size of each ploidy class, i.e., diploid, tetraploid, and octaploid nuclei. After the size interval for each of the ploidy classes was determined, the method allowed determination of ploidy distributions in mouse liver by stereological image analysis alone. The method was established from combined stereological and flow cytometric measurements on liver tissue representing two different stages of liver regeneration after two-thirds partial hepatectomy, and it was tested against an independent set of data representing a marked increase in the portion of S-phase cells.     

A study of hyperlipidaemia induced by ascites tumours.

In mice four stages of hyperlipidaemia induced by Ehrlich ascites tumour could be distinguished. Hyperlipidaemia is characterized mainly by increased serum VLDL content accompanied by high triglyceride concentration. The only exception was the regressive stage III where the serum lipid level (VLDL) has temporarily decreased. From the results obtained with the simultaneous examination of the liver, mesenteric fat tissue, tumour cell and ascites plasma lipids, it may be concluded that the endogeneous fat mobilization induced by tumour cells via increased VLDL synthesis and secretion of liver will lead to hyperlipidaemia and to the total depletion of the lipid stores. Rapid and reversible fall in lipid level following the withdrawal of ascites fluid in tumorous animals demonstrated clearly the direct effect of the tumour cells on lipid-lipoprotein metabolism of the host organism.     

Comparative study of chromosomal nonhistone proteins pp23 in cells from different types of tissue]

A specific immunoserum against nonhistone chromosomal protein pp23 from rat kidney was produced. A similar protein was obtained in cells of different tissue specificity (in the liver cells of rats submitted to a single effect of hepatocarcinogens, in rat hepatomas G-27, HTC, Zajdela, in cultivated non-tumor myogenic L6 cells and in kidney cells of newborn rats) by means of immunofluorescent method on using this immunoserum. In all cases a specific fluorescence of the nucleus and nuclear membrane was observed.     

Microspectrophotometry of cell nuclei stained with the Feulgen reaction. IV. Formation of tetraploid nuclei in rat liver cells during postnatal growth

1. DNA contents of the individual parenchymal nuclei of rat livers during postnatal growth were estimated by microspectrophotometric apparatus, and different ploidy classes of nuclei were classified by their DNA contents. With the same material the total number of parenchymal nuclei in the liver was counted microscopically. 2. If the DNA content of nuclei encountered most frequently in several tissues represents the diploid class, the ploidy classes of the rat liver cell nuclei correspond to di-, tri-, tetra-, and octoploid, with the di- and tetraploid ones predominating considerably. 3. In suckling rats (below 25 gm. of body weight) the liver parenchyma is composed almost exclusively of cells with diploid nuclei, whereas in young rats (above 80 gm.), of tetraploid nuclei. In the growth stage between 25 and 80 gm., there is a remarkable replacement of the diploid nuclei by the tetraploid ones. However, in the liver of adult rats weighing more than 150 gm., any increase or decrease in the frequency of diploid and tetraploid nuclei is hardly observable. In such rats, the nuclear population of the liver parenchyma seems to reach a cell-ecological equilibrium which is considered to be a stable one. 4. It is shown that such nuclear populations and the total number of nuclei in a liver are controlled by the growth state, and not by the age. 5. The decrease in the total number of diploid nuclei and the increase in tetraploid nuclei in the growing livers of rats weighing from 40 up to 130 gm. can both be explained by the hypothesis that the tetraploid nuclei originate from the interphase diploid nuclei without involving mitosis. This hypothesis implies that mitosis is confined to the reproduction of diploid cells alone. 6. It is suggested that, in general, the synthesis of DNA does not necessarily result in the formation of visible mitotic chromosomes. 7. Mitotic time and generation time of diploid nuclei and the percentage of the tetraploidization from diploid nuclei are calculated and discussed.     

Metakaryotic stem cell nuclei use pangenomic dsRNA/DNA intermediates in genome replication and segregation

Bell shaped nuclei of metakaryotic cells double their DNA content during and after symmetric and asymmetric amitotic fissions rather than in the separate, pre-mitotic S-phase of eukaryotic cells. A parsimonious hypothesis was tested that the two anti-parallel strands of each chromatid DNA helix were first segregated as ssDNA-containing complexes into sister nuclei then copied to recreate a dsDNA genome. Metakaryotic nuclei that were treated during amitosis with RNase A and stained with acridine orange or fluorescent antibody to ssDNA revealed large amounts of ssDNA. Without RNase treatment metakaryotic nuclei in amitosis stained strongly with an antibody complex specific to dsRNA/DNA. Images of amitotic figures co-stained with dsRNA/DNA antibody and DAPI indicated that the entire interphase dsDNA genome (B-form helices) was transformed into two dsRNA/DNA genomes (A-form helices) that were segregated in the daughter cell nuclei then retransformed into dsDNA. As this process segregates DNA strands of opposite polarity in sister cells it hypothetically offers a sequential switching mechanism within the diverging stem cell lineages of development.