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PubMed1975

Polypeptide composition of fraction I protein from Nicotiana glauca and from cultivars of Nicotiana tabacum, including a male sterile line.

The polypeptide compositions of fraction I protein isolated from six collections of Nicotiana glauca and from ten cultivars of N. tabacum, as well as a polyploid series and a male sterile line, have been analyzed by isoelectric focusing in polyacrylamide gel slabs containing 8 M urea. Apart from the male sterile line, none of the plants showed any variation from the species pattern of polypeptides. Fraction I protein from the male sterile Burley 21 cultivar of N. tabacum contained the two small subunit polypeptides of N. tabacum and the three large subunit polypeptides of an Australian species, probably N. megalosiphon. This indicates a changed chloroplast genome in the male sterile line in comparison to the normal fertile N. tabacum.

Genetic Variation↗
PubMed1976

Chromosome number and flavonoid synthesis in Briza L. (Gramineae).

Following a recent reidentification of the apigenin C-glycosides in diploid Briza media, the luteolin C-glycosides were reexamined and three acylated derivatives of a luteolin C-glycoside were found. In an attempt to identify the chromosome or group of chromosomes responsible for the change in flavonoid synthesis from 4'-hydroxy- in diploid plants to 3',4'-dihydroxyflavone C-glycosides in autotetraploid plants, leaf flavonoids of artificially produced aneuploids of B. media were examined. Among these plants three different leaf flavonoid profiles were recognized: diploid, tetraploid, and a "modified" tetraploid pattern. All the aneuploids with the normal or "modified" tetraploid pattern were trisomic for one of the small acrocentric chromosomes. Induced polyploids of other Briza species were usually found to have similar flavonoid patterns in the two chromosome races. Flavonoid sulfates were found in three South American species but are absent from all the European species.

Aneuploidy↗
PubMed1975

Cytochemistry of the differentiating flight muscles of the desert locust Schistocerca gregaria.

Cytochemical changes in the metathoracic dorsal longitudinal flight muscle of the female desert locust, Schistocerca gregaria, during its differentiation from the last day of the fourth larval instar, through the fifth larval instar up to the eighth day after the imaginal moult were examined with respect to: (1) DNA content, (2) transcriptional activity, (3) degree of chromatin condensation, (4) RNA content, (5) protein content and (6) cross-secetional fibre area. Microspectrophotometric analysis of Feulgen-stained nuclei shows that polyploidization is not a phenomenon associated with the differentiation of these muscles. The nuclei remain diploid throughout the developmental period. By "staining" with 3H-Actinomycin D, followed by autoradiography, the changes in transcriptional activity coincedent with flight muscle development were examined. At the beginning of the developmental period the transcriptional activity is relatively high and remains so up until about the middle of the fifth instar after which time it declines fairly steadily. These changes in transcriptional activity are accompanied by changes in the degree of chromatin condensation. Generally, the periods of high transcriptional activity exhibit less condensation of the chromatin. The originally high transcriptional activity is accompanied by a steady over-all increase in total RNA content during the developmental period; and in turn a large increase in total protein content. The cross-sectional area of the muscle fibres increases significantly during the growth and differentiation of the flight muscles.

Animals↗
PubMed1982

Nuclear changes accompanying cell differentiation in stems of Pisum sativum L.

Nuclear DNA, nuclear protein and nuclear size have been measured in cells of the cortex, pith and vascular tissue from three successive internodes in the stem of Pisum sativum. New techniques of computer-linked cytophotometry were used to measure these parameters simultaneously in both section and squash preparations. In cortical cells no endoreduplicated nuclei were seen in the internodes measured. In cortical cells from the oldest internode measured, a population of large nuclei with the 2C DNA amount was observed which was not present in the younger internodes. In the oldest pith nuclei measured a few 8C nuclei were present, but maturing pith was most characterized by increasing nuclear size and the population of nuclei accumulating with the 4C DNA amount. Polyploid nuclei were present in all of the vascular tissue measured, including the youngest internode. Maturing vascular tissue was also characterized by increasing nuclear size. Nuclear protein measurements demonstrated a close link between nuclear protein and nuclear size and suggest that increased nuclear size, with constant DNA content, may be due to increased nuclear protein. This raises the question of the nature and function of this nuclear protein, perhaps more characteristic of differentiating cells than dividing cells.

Cell Differentiation↗
PubMed1981

Microfluorometric investigations of chromatin structure. I. Evaluation of nine DNA-specific fluorochromes as probes of chromatin organization.

The ability of the highly condensed chromatin of small thymocyte nuclei and the more loosely organized chromatin of hepatocyte nuclei to interact with nine DNA-specific fluorochromes was assessed by microfluorometry. Although the results obtained with five of the fluorochromes - mithramycin, 7-aminoactinomycin D, Hoechst 33258, DAPI, and propidium iodide - were found to be virtually unaffected by differences in the degree of condensation of the chromatin, the values obtained with the remaining fluorochromes - proflavine, quinacrine mustard, berberine sulfate, and pyronin Y - appeared to be affected significantly by organizational differences of the chromatin. All of the latter "structural probes," except quinacrine mustard, produced fluorescence values which were higher in the 2c nuclei of hepatocytes than in the nuclei of small thymocytes. Quinacrine mustard yielded higher values in thymocyte nuclei; and in the hepatocyte polyploid series (2, 4, and 8c), it did not produce the expected multiples of the 2c value. Pretreatment of the two types of nuclei with RNase affected their total fluorescence in unpredictable ways. While RNase extraction lessened the differences between thymocyte and 2c hepatocyte nuclei stained with propidium iodide, Hoechst 33258, proflavine, and berberine sulfate, it increased the differences between nuclei stained with mithramycin, quinacrine mustard, pyronin Y, and 7-aminoactinomycin D. The ability of RNA-depleted chromatin to interact with various types of fluorochromes might be a useful parameter in subsequent studies of chromatin organization.

Animals↗
PubMed1985

Determination of DNA content in the nurse and follicle cells from wild type and mutant Drosophila melanogaster by DNA-Feulgen cytophotometry.

DNA replication patterns in the nurse and follicle cells of wild type and a female sterile mutant, fs(1)1304, of Drosophila melanogaster have been studied by DNA-Feulgen cytophotometry, using a cell dispersal technique that allowed the measurement of DNA amounts in individual nuclei from egg chambers of known developmental stages. DNA-Feulgen values associated with various ovarian nuclei from egg chambers at different stages of development were used to assess a base line DNA content for ovarian tissues and to estimate the extent of DNA replication in the nurse cells and follicle cells of growing and mature egg chambers. Our data show that both the nurse and follicle cells undergo multiple cycles of endonuclear DNA replication and that there may be selective amplification as well as underreplication by portions of the genome in these highly polyploid, ovarian cells. Alternative models are proposed to account for the DNA replication patterns observed. Comparisons of DNA-Feulgen levels in wild type ovarian nuclei with those found for the fs(1)1304 mutant and its heterozygote in the balanced stock fs/FM3, show that equivalent DNA levels are present in follicle cell nuclei from all three types of females. Nurse cell nuclei in the homozygous fs stock, however, fail to achieve the same high DNA levels observed in both fs/FM3 and wild type nurse cell nuclei. Although the nuclei of follicle cells in ovaries from fs/fs females appear morphologically like those surrounding egg chambers in wild type ovaries, nurse cell nuclei from mutant females show a more compacted organization of their chromatin than found for nurse cell nuclei from wild type ovaries at similar developmental stages. Our findings suggest that a major effect of the fs(1)1304 mutation may be on the coiling behavior of chromatin and the conformation of DNA-protein moieties in both nurse cell and follicle cell nuclei. These changes in chromatin structure apparently are manifest by perturbations in DNA replication patterns and normal gene function in these biosynthetically active cells.

Animals↗
PubMed1981

DNA distribution of mast cell populations in growing rats.

The proliferation of rat peritoneal mast cells was examined under normal conditions in vivo. DNA content of individual mast cells was measured by cytofluorometry after staining with the bibenzimidazole dye Hoechst 33258. Diploid non mast cells from each rat were used as a biological standard, which resulted in small long-term variations in the method. The proportion of mast cells in the S + G2 region of the DNA distribution was about 4% for young rats (24 days old, body-weights about 60 g). It decreased in relation to body-weight, and was less than 1% for 105-day-old rats weighing 400 g. During the same growth period the total number of mast cells in the peritoneal cavity increased about 8-fold. The total number of proliferating cells, about 30,000, remained constant throughout the observation period. No evidence of polyploidization or accumulation in G2 of mast cell nuclei was found. It is concluded that peritoneal mast cells increase in number by mitotic proliferation of differentiated cells.

Animals↗
PubMed1987

Serial section method for cytofluorometric determinations of the DNA content of component cells of the human cochlea.

We describe a method for measuring the DNA content of the component cells of the organ of Corti using serial sections of human cochleae obtained at autopsy. Cochleae were fixed in Carnoy's solution and embedded in Acrytron E, a water-miscible methacrylate resin. A procedure was developed to reduce the background fluorescence in methacrylate-embedded sections; the resin was pretreated with ion-exchange resin (Amberlite IRA-410). Experiments showed that pretreatment reduce the background fluorescence practically to zero. Seventy 3 microns-thick serial sections were prepared on fluorescence free glass slides and stained with azocarmin G and acriflavine-Feulgen. After postirradiation using blue excitation light, the amount of Feulgen-DNA present in the target nucleus in each section was determined using a microfluorometer. The amount of DNA in the entire nucleus was determined by adding together the DNA content of the segments of the nucleus. The characteristic appearance of the organ of Corti made it easy to detect these cells; under green excitation light the cells of this organ exhibited red cytoplasmic azocarmin-G fluorescence. Due to the relatively wide internuclear spaces, cytofluorometry fo individual nuclei could be performed without interference from the neighboring cells. Our technique using serial sections allowed us to measure the DNA content of individual cells and obtain histological information about particular cells and their neighboring cells. Several polyploid cells were found among the Hensen's cells in the cochlea, while all other component cells of the organ of Corti were diploid.

Cell Nucleus↗
PubMed1991

Karyotypic stability of serum-free mouse embryo (SFME) cells.

Mouse embryo cultures derived in serum-containing medium undergo growth crisis or senescence after fewer than 20 population doublings, followed by the emergence of genetically altered, polyploid 'immortalized' cells capable of growing indefinitely. Serum-free mouse embryo (SFME) cells, derived in medium in which serum is replaced with growth factors and other supplements, do not exhibit growth crisis or gross chromosomal aberrations when cultured for well over 100 population doublings and display other unique properties. We examined culture conditions and physiological factors affecting karyotypic stability in long term cultures of SFME cells derived from several mouse strains. Cloning SFME cells consistently isolated colonies with altered karyotype, even when the clones were derived from parent cultures with no karyotypic alterations. After 140-200 population doublings in vitro, the percentage of SFME cells showing hyperdiploidy or structural chromosomal abnormalities increased, although the modal chromosome number remained diploid. SFME cells transformed with molecularly cloned oncogenes did not show alterations in karyotype beyond that expected from the clonal origins of these cells, indicating that malignant transformation of SFME cells does not result in general karyotypic instability.

Animals↗
PubMed1985

Bradykinin causes a transient rise of intracellular Ca2+-activity in cultured neural cells.

The concentration of intracellular free calcium ions was measured by spectrofluorometry in suspensions of quin2 loaded neural cell lines: neuroblastoma X glioma hybrid cells (clones 108CC15 and 108CC25) and polyploid rat glioma cells (clone C6-4-2). In these cells, bradykinin elicits a transient increase of the cytosolic Ca2+-activity in a dose-dependent manner (half-maximal effect at about 10 nM). The effect requires the presence of extracellular Ca2+. The time to peak is at most 10 s, the decay to the original level lasts 1 min and is followed by a period of 1-4 min during which Ca2+ activity is slightly below control value. Lys-bradykinin and Met-Lys-bradykinin evoke similar effects as bradykinin, but at concentrations 10 times lower. The cells desensitize upon repeated addition of bradykinin. Under the same conditions des-Arg1-bradykinin, des-Arg9-bradykinin, angiotensin II, substance P, apamin and histamine exerted no influence on the concentrations of free Ca2+. Similar to their effect in neural cell lines, bradykinin and Lys-bradykinin induce in primary astroglia-rich cultures from rat brain an increase in the concentration of cytosolic Ca2+ with the peak reached within 30 s and the decay to the original level lasting approximately 4 min. The significance of this effect of bradykinin on the cytosolic Ca2+-activity is discussed in relation to previous findings that bradykinin in the same cell lines induces a hyperpolarization, a rise of the cyclic GMP level and a breakdown of phosphoinositides.

Aminoquinolines↗
PubMed1982

DNA-flow-cytometric measurements on the normal, atrophic, hyperplastic and neoplastic human endometrium.

DNA distribution patterns and the fractions of the cell cycle phases were determined by means of flow-through cytometry in 87 samples of normal, atrophic, hyperplastic and carcinomatous human endometrium. The S-phase fractions vary during the normal menstrual cycle between 1 and 3% and reach a periovulatory maximum between 4.4 and 4.7%. Atrophic endometrium and regressive glandular cystic hyperplasia have little DNA synthesis (1.01% and 1.68% S-phase fractions respectively). Proliferating glandular cystic hyperplasia reveals 3.38% S-phase fraction, whereas adenomatous hyperplasia has an increased number of DNA-synthesizing cells (4.81%). The well-differentiated endometrial carcinoma shows no cytophotometrically detectable differences in comparison to adenomatous hyperplasia. All endometrial samples except for poorly differentiated endometrial carcinoma showed a diploid to tetraploid DNA distribution pattern. The poorly differentiated endometrial carcinoma displays two different types: one rapidly growing diploid-tetraploid tumor with 8.0 to 9.6% S-phase fractions, and another type with stemline deviations, polyploid nuclei and less pronounced synthetic activity.

Adenoma↗
PubMed1979

Flow cytometric analysis of the DNA distribution in human brain tumors.

Flow cytofluorometric analysis was used to determine the distribution of the DNA content in cells from selected areas of normal human brain and in benign and malignant brain tumors. Propidium iodide was employed as DNA fluorochrome and the analysis was carried out on a suspension of single cells. Normal, nonstimulated human lymphocytes were used as diploid controls. With nonneoplastic tissue an average of 91% of the cells were diploid (presumably in G0 or G1 stage of the cell cycle). The cells of most benign tumors were mainly diploid (77-98%), nine specimens of pituitary adenomas had large numbers of aneuploid cells. In glioblastoma multiforme the proportion of diploid cells was significantly diminished and polyploid cells were frequently seen. Similar results were obtained in other malignant tumors, with metastatic tumors showing the greatest ploidy variation, which included triploid, tetraploid, and hypertetraploid cells. The analytical method used provides valuable information of significant clinical importance on the DNA distribution in brain tumor cells.

Aneuploidy↗
PubMed1995

Antimicrotubule effects of the novel antitumor benzoylphenylurea derivative HO-221.

The antitumor action of HO-221, a novel benzoylphenylurea derivative, was studied. The in vitro cytotoxic strength of HO-221 was investigated, as measured by IC50 values, compared with those of other drugs with different action mechanisms, using Chinese hamster lung (CHL) cells, mouse leukemia L1210 cells and human promyelocytic leukemia HL-60 cells. Morphological alterations following treatment were observed under a phase contrast microscope, and the mitotic index was determined at regular intervals to check for accumulation of metaphase cells. HO-221 was found to have a very strong toxic effect on all cell types, equal to that of the spindle poisons used as controls. HO-221 also produced the same specific morphological changes as the spindle poisons, with a significant accumulation of metaphase cells. A chromosome analysis of treated cells showed that HO-221 frequently induced polyploid and aneuploid cells, but without accompanying chromosome-breaking activity. An in vivo mouse bone marrow micronucleus assay was also carried out. The assay allowed the in vivo identification of a chromosome breaker or a spindle poison through the measurement of the relative sizes of micronuclei produced and erythrocytes. HO-221 was found frequently to induce relatively large micronuclei, an action regarded as specific to spindle poisons. It was thus demonstrated that HO-221 acts as a spindle poison both in vitro and in vivo. In order to investigate the mechanism of this action, a study of tubulin assembly using purified calf brain tubulin was carried out, which demonstrated clearly that HO-221 inhibits microtubule assembly. A detailed investigation of the action mechanism of HO-221 as a spindle poison is now called for.

Animals↗
PubMed1993

Cell kill kinetics and cell cycle effects of taxol on human and hamster ovarian cell lines.

Taxol is a clinically active anticancer drug, which exerts its cytotoxicity by the unique mechanism of polymerizing tubulin monomers into microtubules and stabilizing microtubules. Our studies with ovarian (hamster CHO and human A2780) cells showed that taxol is a phase-specific agent that is much more cytotoxic to mitotic cells than interphase cells. First, the dose-survival pattern of taxol resembled that of other phase-specific agents, in which cell-kill reached a plateau at a certain concentration. This suggests that the asynchronous cell population consists of a taxol-sensitive (presumably mitotic) fraction and a taxol-resistant fraction. Second, the cells were more responsive to increased exposure time than to increased dose above the plateau concentration. Third, in both asynchronous and synchronous cultures taxol was much more cytotoxic to mitotic than interphase (G1, S and G2) cells. Fourth, the taxol concentration needed to kill cells corresponded to the dose needed to block cells in mitosis. Although taxol blocked cells in mitosis, the mitotic block was of short duration. Cells escaped the mitotic block, without cytokinesis, and entered the next round of DNA synthesis to form multinucleated polyploid cells. Taxol was 15- to 25-fold more toxic to A2780 (human ovarian carcinoma) cells compared to CHO cells. This difference in sensitivity correlated with a higher intracellular taxol concentration in A2780 as compared to CHO as determined by either an ELISA assay or by [H3]-taxol uptake.

Animals↗
PubMed1994

Accumulation of HL-60 leukemia cells in G2/M and inhibition of cytokinesis caused by two marine compounds, bistratene A and cycloxazoline.

The effects on the cell cycle of two biologically active compounds, bistratene A and cycloxazoline, from the marine ascidian Lissoclinum bistratum were studied in HL-60 human leukemia cells using flow cytometry. Both compounds were shown to cause an apparent accumulation of cells in the G2/M phase. This effect was shown to be both time- and dose-dependent. At the longer time points (30 and 48 h after addition of the compounds) polyploidy was apparent. The fate of cells labeled in the S phase with 5-bromo-2'-deoxyuridine (BrdUrd) was analysed using a bivariate BrdUrd/PI (propidium iodide) technique. Bistratene A and cycloxazoline treatment prevented the majority of BrdUrd-labeled cells from progressing through to the G1 phase. Approximately 50% of the cells were delayed at G2/M, and a significant proportion of cells appeared to be polyploid. Light and electron microscopy revealed the presence of multinucleated cells accounting for the apparent polyploidy. The progression of cells out of the G1 phase was also examined by synchronising cells with mimosine and releasing them from mimosine block in the presence of bistratene A. There was no evidence of a block at the G1/S phase transition or through the S phase since DNA synthesis was not inhibited. The mechanism by which these compounds interfere with cytokinesis is presently unknown but, in the case of bistratene A, may be linked to altered phosphorylation of cellular proteins involved in cell-cycle control.

Acetamides↗
PubMed1979

Iron-induced atypical meningioma cells?

Polymorphous tumor cells with bizarre giant nuclei are characteristic of the so-called atypical meningioma. Some of these cells are iron-positive. Meningioma cells apparently are able to store iron excessively. It is discussed whether an extremely high intracellular iron uptake is responsible for atypical meningioma growth. Derangement of mitotic spindles may lead to polyploid chromosome sets and thereby to giant nuclei. This seems likely because induction of tumor growth after iron injection has been described. Spodographic examinations showed that even histologically inconspicuous tumor cells contain fine-grained iron. Application of the Prussian-blue reaction to spodograms reveals distinctly higher iron contents than common paraffin sections had proved.

Cell Nucleus↗
PubMed1995

Genome reorganization from polyteny to polyploidy in the nurse cells found in onion fly (Delia antiqua) and cabbage root fly (Delia radicum) ovaries (Diptera, Anthomyiidae).

The material required to ensure successful embryogenesis in the onion fly (Delia antiqua) and the cabbage root fly (Delia radicum) (Diptera, Anthomyiidae) is supplied by 15 nurse cells, while the oocyte chromosomes enter a quiescent stage during prophase I of meiosis. This level of transcription is achieved by the polyploidization of the nurse cell DNA. Elongate polytene chromosomes form in both species, but lack the banding and conspicuous puffing commonly seen in other dipteran tissues. The polytene chromosomes contract until they finally appear as small, densely staining spheres. These fragment into large numbers of endochromosomes that are much smaller than their mitotic counterparts, which then despiralize, resulting in the flocculate appearance of the nurse cell nucleus. Photodensitometry revealed a gradient of DNA values between nurse cells near the oocytes and those further away. Final DNA values 1000 times the haploid level were recorded in the nurse cell nearest to the oocyte compared with 336 times the C-value in the most distal cell. At lower temperatures (< 10 degrees C), the polytene chromosomes become banded and longer. None of the onion flies kept in these conditions produced viable eggs, though there was some reproductive success among the cabbage root flies.

Animals↗