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D Schroeter

Publications and source records attributed to D Schroeter.

35 records · Page 2Linked to original sources
PubMed1991

Derangement of microtubule arrays in interphase and mitotic PtK2 cells treated with deuterium oxide (heavy water).

The extent and pattern of the rearrangements of microtubule arrays in interphase and mitotic PtK2 cells treated with deuterium oxide (2H2O) were evaluated using light, immunofluorescence and electron microscopy. Combined labelling with anti-tubulin antibodies and staining with a DNA-specific fluorochrome revealed that 2H2O influences the reassembly of the cytoplasmic microtubule complex (CMTC) of interphase cells after depolymerization of microtubules (MTs) with nocodazole. In cells entering mitosis in the presence of 75% 2H2O the conversion of the CMTC into the mitotic spindle was affected, resulting in a retardation of the prophase/prometaphase transition. (Pro)metaphase cells did not assemble a regular mitotic spindle and the metaphase/anaphase transition was blocked. Immunofluorescence and ultrastructural studies suggest that separation of centrosomes, nucleation of MTs around centrosomes, organization of MTs into the mitotic spindle, as well as the ultrastructure and positioning of the mitotic poles, are affected in deuterated PtK2 cells. In comparison with control cells, a significantly higher proportion of multipolar divisions was found after stimulation of proliferation in the presence of 25-50% 2H2O or during recovery after a long-term exposure to 75% 2H2O. On the basis of these results we discuss the mechanism of the antimitotic action of deuterium oxide and suggest that, apart from perturbation of MT polymerization, it could also encompass disturbances in MT reorganization, most probably by impairment of the microtubule-organizing centres (MTOC).

Animals↗
PubMed1990

Centromere separation. Early replication of repetitive DNA associated with inactive centromeres.

Four types of stable dicentric and one octacentric chromosomes from mouse brain tumor cells and L-929 cells were analyzed for the timing of replication of repetitive deoxyribonucleic acid (DNA) located in the centric and pericentric regions associated with active versus inactive centromeres. The repetitive DNA present in the heterochromatin blocks of inactive centromeres replicates much earlier than similar DNA associated with the active centromeres. The former appears to replicate during early to mid S when several euchromatic segments are still replicating. There seems to be little or no overlap in the timing of replication of the repetitive DNA present in the vicinity of prematurely separating centromeres (which are accessory and nonfunctional) and those that separate at meta-anaphase junction (which are the functional centromeres). In the absence of any information about the mechanism(s) controlling initiation and completion of DNA synthesis in the two types of heterochromatic blocks, the differential timing of replication of the DNA with similar base composition remains an enigma.

Animals↗
PubMed1990

Mitosis arrested by deuterium oxide. Light microscopic, immunofluorescence and ultrastructural characterization.

Dynamics and correctness of mitotic division of PtK1 cells grown in vitro in the presence of deuterium oxide were assessed by morphological criteria. Incubation of PtK1 monolayers for 2 h in media supplemented with 25% to 75% D2O retarded in a dose-dependent manner the interphase/prophase transition, thus substantially decreasing the fraction of prophase cells. The progression of mitosis was also arrested at the metaphase/anaphase transition resulting in an accumulation of prometaphase and metaphase cells. After a prolonged incubation (12-24 h) in 75% D2O, prophase cells reappeared, often displaying a peculiar, "overmatured" pattern of chromosome condensation. In the presence of D2O an "overmatured" prophase cell is able to transform into a prometaphase cell and then directly into a multinucleate/micronucleate cell. Arrested prometaphase and metaphase cells developed restitution nuclei when incubated up to 24 h in the media containing 25% to 75% of heavy water. Immunofluorescence and electron microscopic studies revealed that the treatment with deuterium oxide profoundly disturbed development of the mitotic spindle. On the basis of these observations, a possible mechanism of the antimitotic action of deuterium oxide is discussed. We postulate that centrosomally controlled transformation of the microtubular cytoskeleton into the mitotic spindle is impaired by D2O. This delays the progression of prophase and disturbs congression of chromosomes into a regular metaphase plate.

Animals↗
PubMed1989

Sequence of centromere separation: kinetochore formation and DNA replication in dicentric chromosomes showing premature centromere separation in rat cerebral cells.

A subpopulation of rat cerebral endothelial cells, designated B1, exhibits an array of multicentric chromosomes. Because of the formation of bridges at anaphase, this cell population produced new types of multicentrics at every cell division. These chromosomes showed kinetochore proteins at every centromeric site and all centromeric regions replicated their DNA at the end of the S phase, more or less simultaneously. A new subpopulation of cells, designated B2, obtained from the original sample frozen at Wayne State University displayed several dicentrics. In contrast to B1 these chromosomes exhibit premature centromere separation as reported for mouse and human cell lines. These B2 dicentrics show only one site of kinetochore protein deposition. The timing of DNA replication around the centric region of prematurely separating centromere is also changed similar to the earlier reported premature DNA synthesis for mouse dicentrics. These observations suggest a universality of relationship between premature centromere separation, a lack of kinetochore formation, and early replication of the centric/pericentric DNA associated with these centromeres. The cause of sudden change from activity to inactivity of these chromosomes, though interesting, is not clear.

Animals↗
PubMed1989

Disorganization of mitosis in HeLa cells by deuterium oxide.

We assessed, by light and electron microscopy, the influence of deuterium oxide on the dynamics of mitosis and on the morphology of the mitotic apparatus in HeLa cells grown in vitro. A 2-h incubation of HeLa monolayers with low concentrations of D2O (1%-25%) in the medium increased the frequency of multipolar divisions up to 20 times the control level. Substitution of 10% and 25% D2O for H2O induced changes in the proportions of mitotic phases. These changes could be fully reversed to the control pattern after 1 h of recovery in non-deuterated medium. Fifty % D2O strongly inhibited, and 75% D2O blocked the cell cycle before prophase and at (pro-)metaphase. In cells treated with 50% D2O conspicuous morphological changes of the interphase chromatin as well as ultrastructural abnormalities of all mitotic phases were regularly observed. Overall, these results confirmed the antimitotic activity of deuterium oxide and revealed that it could also influence the cell cycle before mitosis. It is suggested that interference with diverse cellular constituents rather than a specific influence on microtubule turnover could be responsible for the disorganization of the cell cycle in HeLa cells by D2O.

Chromatin↗
PubMed1986

Preparation of two plasma membrane fractions from ascites tumour cells by gel chromatography on Sephacryl S-1000.

Two plasma membrane fractions from ascites tumour cells with differences in vesicle size were isolated by gel-exclusion chromatography on Sephacryl S-1000. Fraction 1 appeared in the void volume and had a vesicle diameter in the range 300-400 nm. Fraction 3, with an equilibrium constant (Kd) of 0.58, consisted of vesicles between 100 and 200 nm in diameter as measured by routine size analysis with the electron microscope and by calibration of the column with latex beads. The appearance of two plasma membrane fractions could also be confirmed by iodination of the surface membrane prior to fractionation. This gel chromatographic procedure represents a rapid and convenient method for the isolation of membrane material, which was enriched between five- and fourteen-fold based on the specific activity of the membrane-bound marker enzymes. Fraction 1 contained small amounts of lysosomal and Golgi membranes, and fraction 3 some material of the Golgi apparatus and the endoplasmic reticulum. The major portion of the contaminating membraneous material remained on the column and could be eluted with high salt buffer. The two plasma membrane fractions revealed some differences in 5'-nucleotidase specific activity and in the protein pattern, especially in the higher molecular weight range, as shown by sodium dodecyl sulphate gel electrophoresis.

Acrylic Resins↗
PubMed1985

Cellular compartments in mitotic cells: ultrahistochemical identification of Golgi elements in PtK-1 cells.

This investigation focuses on the identification, distribution, and transport of intracellular membrane systems during mitosis. The membranes of the Golgi apparatus can be identified cytochemically by staining for acid phosphatase (acPase) and thiamine pyrophosphatase (TPPase) activity. Using this approach we are able to study the disintegration of the Golgi apparatus during mitosis and to follow the dislocation as well as the organized reappearance of Golgi elements after the completion of mitosis. We are able to demonstrate that during mitosis the activity of both enzymes is strong enough to react with the substrate applied during the staining procedure. Furthermore, we observe a characteristic pattern of membrane distribution in mitotic cells. During interphase the TPPase reaction is characteristically limited to one or two cisternae of a dictyosomal stack. The acPase reaction stains the membranes of the total stack, of the GERL, of some vesicles and cisternae near the dictyosomes and lysosomes. After the mitotic breakdown of the dictyosomal stacks the forming vesicles still stain positively and are distributed over the entire cytoplasm. At late anaphase and early telophase the enzyme activity occurs not only in the reconstituting dictyosomes but also in the nuclear envelope and in some ER cisternae. The extended spectrum of membrane structures indicating Golgi enzyme activity becomes obvious. This phenomenon favors the idea that at least some functions of the Golgi apparatus persist during mitosis.

Acid Phosphatase↗
PubMed1983

In vitro formation of different tubulin polymers from purified tubulin of Ehrlich ascites tumor cells.

Preparations of cycled tubulin from Ehrlich ascites tumor cells contain several accessory proteins; once or twice cycled microtubule preparations are usually composed of fibers 10 nm in diameter, but lack vimentin. Highly purified tubulin consists of alpha- and beta-tubulin and a minor component which was identified by peptide mapping as a second beta-chain. This pure tubulin is able to form in vitro at low concentrations (1 mg protein/ml) fibers of about 10 nm width, and at higher concentrations (3.5 mg protein/ml) normal microtubules.

Animals↗
PubMed1983

Pig sperm tail tubulin. Its extraction and characterization.

Axonemal tubulin extracted from pig sperm tails has been characterized by one- and two-dimensional electrophoresis and by one-dimensional peptide mapping. The electrophoretic mobilities of its subunits after reduction and carboxymethylation were similar to those of the major subunits of pig brain tubulin. Sperm tail tubulin subunits also had roughly the same isoelectric points as pig brain tubulin subunits, except that they appeared to have a relatively larger tailing effect. The proteolytic cleavage pattern of the pig sperm tail beta-tubulin closely resembled those of both the tunicate (Ciona intestinalis) sperm beta-tubulin and pig brain beta-tubulin. The peptide pattern of pig sperm tail alpha-tubulin, however, was more similar to that of tunicate sperm tail alpha-tubulin than to that of pig brain alpha-tubulin. This supports the hypothesis put forward in a previous investigation [1] that functionally similar tubulins from taxonomically distant species can be more related than functionally dissimilar tubulins from the same species.

Animals↗
PubMed1982

[Not Available].

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Demography↗
PubMed1981

Size of native and denatured DNA of Ehrlich ascites tumour cells isolated in the presence of different protease concentrations.

Native DNA molecules isolated either in the presence of 50 micrograms x ml(-1) of proteinase K (PK-DNA) or in the presence of 6 mg x ml(-1) autodigested pronase (PRO-DNA) are about equal in size. Since shear forces were avoided as far as possible during the isolation procedure, the largest molecules found were longer than 100 microns. The average length of the traced molecules was 34.2 microns for PK-DNA and 29.7 microns for PRO-DNA. In contrast to PK-DNA the length of PRO-DNA molecules undergoes a dramatic change during denaturation. The average contour length of a denatured PRO-DNA molecules is only 6.9 microns. This reduction in length cannot be explained by shrinkage due to changes in ionic strength, pH and the effect of denaturing agents. Moreover, PK-DNA identically denatured was not dramatically changed in size. From this it must be concluded that PRO-DNA contains more internal ends than PK-DNA. This conclusion is supported by the results indicating that PRO-DNA is much more sensitive to nuclease S1 than PK-DNA. The results are consistent with previously published biochemical data suggesting that chromosomal DNA is 'nicked' or 'gapped' in a protease-catalyzed reaction at distinct protease-sensitive sites.

Animals↗
PubMed1980

Plasma membrane heterogeneity in ascites tumor cells. Isolation of a light and a heavy membrane fraction of the glycogen-free Ehrlich-Lettré substrain.

In this work we report on the isolation of two plasma membrane fractions of a glycogen-free substrain of Ehrlich-Lettré ascites cells, a light fraction sedimenting in a sucrose gradient at 1.10 g/ml, and a heavy fraction sedimenting at nuclei by a combination of short-term swelling and mild Dounce homogenization. A 12 000 X g postnuclear pellet (PII) containing major portions of the plasma membrane marker enymes, 5'-nucleotidase, ouabain-sensitive (Na+ + K+)-ATPase and the alkaline phosphatase, was prepared by differential centrifugation. The two plasma membrane fractions were obtained by centrifugation on a discontinuous sucrose gradient, from which they were further purified on a linear sucrose gradient applying sedimentation velocity conditions only. Enrichment factors for the three marker enzymes were between 5- and 14-fold for the light fraction and between 3- and 7-fold for the heavy fraction with an overall yield of 1--4% and 0.5--1.7%, respectively, of cellular protein. Contamination of both fractions with nuclear material was minor. Mitochondrial contamination was about 8% for the light material and somewhat higher for the heavy material. In the light fraction, co-sedimentation of lysosomal and Golgi marker enzymes was detected. The presence of membrane structures of these organelles could not be confirmed definitely by electron microscopy. Differences in sialic acid content and phospholipid composition within the two fractions, especially in the relative proportion of lecithin to sphingomyelin, suggests differences in membrane fluidity. The light material showed mostly unit membrane vesicles in thin-section and freeze-etch electron microscopy, whereas the heavy fraction mainly consisted of sheet-like membrane fragments.

5'-Nucleotidase↗
PubMed1979

Assembly of nonneural microtubules in the absence of glycerol and microtubule-associated proteins.

Microtubule protein from Ehrlich ascites tumor cells purified by an in vitro polymerization process in the absence of glycerol and calcium chelators contains several accessory proteins but lacks the high molecular weight proteins which are present in neurotubulin. DEAE-Sephadex chromatography of two-times cycled tubulin removes these nontubulin proteins, resulting in pure tubulin, as critically examined by sodium dodecyl sulfate gel electrophoresis. This tubulin can readily assemble into microtubules in assembly buffer, at low magnesium concentrations, without glycerol and at tubulin concentrations above 0.8 mg/mL. Electron microscopy shows that the tubules are identical with normal microtubules. When the purified tubulin fraction was reduced and carboxymethylated, a significant minor protein component could be observed electrophoretically, migrating between alpha- and beta-tubulin. At present, the identity and function of this protein are not known. The results demonstrate that the in vitro assembly of tubulin from Ehrlich ascites tumor cells does not require high molecular weight proteins or gamma-like factor(s) as has been proposed for the neurotubulin system.

Animals↗
PubMed1977

[The influence of beta-sympathomimetics and so-called Ca++-antagonistic inhibitors on human heart muscle in vitro (author's transl)].

In an in vitro assay on fetal human heart muscle it was demonstrated for the first time that overstimulation by beta-sympathomimetics could cause elective parenchymal necrosis. Fenoterolhydrobromide, which is used for tocolysis on a longterm scale, induces in vitro necroses of individual heart muscle fibers according to a pathogenetic principle postulated by Flekkenstein. The combination of Fenoterolhydrobromide with a Ca++-antagonist prevents elective parenchymal necroses by reducing the Ca++-influx into the heart muscle fibers. These results suggest that elective necroses of heart muscle fibers may be not only of coronarogenic but also of metabolic origin.

Calcium↗
PubMed1976

The effects of betamimetic drugs used for tocolysis on the fetal myocardium.

During the last five years tocolysis with betamimetic drugs, e.g. Fenoterol has become a standard method for the treatment of impending premature births. We studied the effect of Fenoterol in vitro on organ cultures of human fetal hearts. At a concentration in the nutrient medium corresponding to the dose used in clinical application, light and electron microscopy demonstrated myocardia damage. In contrast the combined use of Fenoterol and the calcium antagonist Verapamil in a ratio of 1:40 showed no pathological findings either clinically or in vitro. The results are documented with light and electron photomicrographs.

Ethanolamines↗
PubMedDate not supplied

Induction of premature mitosis in cells blocked in S-phase: possibilities of its use in cancer therapy.

Oncogenesis leads to weak regulations of many cell cycle checkpoints. This could be useful for cancer therapy. To test this assumption, five human malignant cell lines were used to induce premature mitosis by overriding the S-phase control, which ultimately leads to cell death. Two cell lines showed a significantly high frequency of premature mitotic cells induced by okadaic acid; whereas the others did not. One cell line, although having a high cyclin B level in cells blocked in S-phase, failed to show premature mitosis. The cyclin B level in cells blocked in S-phase alone could not explain the differential behavior of the cell lines. It was observed that in addition, the activation of cdc25 in these cells could also play an important role in the induction of premature mitosis in such cells.

Cell Adhesion↗