A genetic system to detect mitotic recombination between repeated chromosomal sequences in Drosophila Schneider line 2 cells

In order to study mitotic homologous recombination in somatic Drosophila melanogaster cells in vitro and to learn more on the question how recombination is influenced by mutagens, a genetic system was developed where spontaneous and drug-induced recombination could be monitored. Two recombination reporter substrates were stably introduced in multiple copies into the genome of established D. melanogaster Schneider line 2 cells: one plasmid (pSB310) contained the 5′ and 3′ deleted neomycin phosphoribosyltransferase alleles neoL and neoR as direct repeats; the other (pSB485) contained similar deletions (lacZL and lacZR) of the β-galactosidase gene (lacZ). Restoration of a functional neo gene upon mitotic recombination between homologous sequences allowed direct selection for the event, whereas recombination in single cells harbouring the integrated lacZ-based reporter plasmid was detected by histochemical staining or flow cytometric analysis (FACS). The neo-based construct in the clonal transgenic cell line 44CD4 showed a spontaneous recombination frequency of 2.9×10⁻⁴, whereas the 485AD1 cell line harbouring the lacZ-based construct exhibited a frequency of 2.8×10⁻⁴. The alkylating agents EMS and MMS and the clastogen mitomycin C were able to induce recombination in the 485AD1 cell line in a dose-dependent manner. The results obtained from these studies suggest that the transgenic cell lines are potentially useful tools for identifying agents which stimulate direct repeat recombination in somatic Drosophila cells.     

The effect of sodium fluoride on DNA synthesis, mitotic indices and chromosomal aberrations in human leukocytes treated with trenimon in vitro

Human leukocyte cultures were set up with Ham's F-10 medium and stimulated with PHA-M. Treatment of the cells in G₁ from 15–20 h with 0.5 × 10⁻⁶ M Trenimon resulted in a considerable cell cycle delay, as measured by [³H]-TdR autoradiography and determination of mitotic indices. Under these conditions only few cells incorporated the tracer at the same time as most cells did in untreated cultures. However, this did not lead to a mitotic activity at the same time as obtained in controls. Most of the treated cells started their DNA synthesis and mitotic activities with a delay of around 20 h, as compared with the controls. Continuous treatment of the cells with 10⁻³ M NaF had no effect on [³H]TdR labelling or mitotic indices in otherwise untreated cultures, but led to an impressive effect on DNA synthesis in Trenimon-treated cultures, without a considerable effect on the mitotic indices. This finding could beexplained as due to a lower alkylation in cellular DNA in the presence of NaF. More cells can start with their DNA synthesis, although they are, like Trenimontreated cultures, incapable of completing it normally. Analyses of the effect of NaF on chromosomes aberrations induced by Trenimon revealed that pre-, simultaneous and post-treatments significantly enhanced the frequency of undamaged mitoses. Continuos fluoride treatment also protected the cells from Trenimon-induced damage, but the effect was not significant, possibly because of heavily damaged mitoses which appeared under these conditions. We interpret our findings as an indication of a real anti-mutagenic activity of NaF.     

The differentiation of normal and transformed human fibroblasts in vitro is influenced by electromagnetic fields

We studied the effect of symmetric, biphasic sinusoidal electromagnetic fields (EMF) (20 Hz, 6 mT) on the differentiation of normal human skin fibroblasts (HH-8), normal human lung fibroblasts (WI38), and SV40-transformed human lung fibroblasts (WI38SV40) in in vitro cultures. Cells were exposed up to 21 days for 2 × 6 h per day to EMF. Normal mitotic human skin and lung fibroblasts could be induced to differentiate into postmitotic cells upon exposure to EMF. Concomitantly, the synthesis of total collagen as well as total cellular protein increased significantly by a factor of 5–13 in EMF-induced postmitotic cells. As analyzed by two-dimensional gel electrophoresis of [³⁵S]methionine-labeled polypeptides, EMF-induced postmitotic cells express the same differentiation-dependent and cell type-specific marker proteins as their spontaneously arising counterparts. In SV40-transformed human lung fibroblasts (cell line WI38SV40) the exposure to EMF induced the differentiation of mitotic WI38SV40 cells into postmitotic and degenerating cells in subpopulations of WI38SV40 cell cultures. Other subpopulations of WI38SV40 cells did not show any effect of EMF on cell proliferation and differentiation. These results indicate that long-term EMF exposure of fibroblasts in vitro induces the differentiation of mitotic to postmitotic cells that are characterized by differentiation-specific proteins and differentiation-dependent enhanced metabolic activities.     

Polypeptide synthesis by mitoplasts isolated from mouse liver

Polypeptide synthesis by mouse liver mitochondria was studied by incubating purified mitoplasts (mitochondria treated with digitonin) with [³⁵S]methionine. The products were separated either by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, or by isoelectric focusing, followed by SDS polyacrylamide gel electrophoresis. At least 14 distinct bands with molecular weights (mol. wt) ranging from about 8 000 to about 70 000 were found upon radioautography of the gels. When the samples were incubated in the presence of chloramphenicol, only a single weak band was found, whereas the protein pattern was unaffected by the presence of cycloheximide in the medium. The newly synthesized proteins were all acidic and evidence was obtained that they were hydrophobic in nature. Virtually all the labelled polypeptides were present in the membrane fraction, whereas the matrix showed little radioactivity. The data indicate that the proteins synthesized by mammalian mitochondria, like those in yeast, are components of the inner mitochondrial membrane. One protein of mol. wt 22 000 D was detected in the incubation medium. Since more of this component was present in the medium than in the pelleted mitoplasts and since this protein was not found in the matrix fraction of sonicated mitoplasts, it is believed that it had been excreted from the inner mitochondrial membrane. The finding that the number of proteins synthesized in mitoplasts isolated from mouse liver is considerably higher than that synthesized in yeast mitochondria reflects a most efficient utilization of the mammalian mitochondrial genome.     

Effect of diphtheria toxin fragment A on energy coupling in mitochondria. Studies on mouse liver mitoplasts

The effect of intact diphtheria toxin and of its fragment A on protein synthesis in mouse liver mitoplasts (digitonin-treated mitochondria) was studied. Fragment A inhibited protein synthesis in intact mitoplasts to the same extent as the uncoupler, carbonylcyanidep-trifluoromethoxyphenylhydrazone, but similar effects were not observed in lyzed mitoplasts. Intact diphtheria toxin was without effect in either case.Fragment A strongly stimulated mitochondrial ATPase activity. At concentrations which efficiently inhibited mitochondrial protein synthesis and stimulated ATPase activity, fragment A had no effect on the intramitochondrial concentration of nicotin-amide adenine dinucleotides. Moreover, it did not catalyze ADP ribosylation of mitochondrial proteins. The results indicate that the effects observed did not involve the NAD⁺-glycohydrolase activity of fragment A.[¹²⁵I]-Labelled fragment A was bound to mitoplasts to about the same extent as the labelled intact diphtheria toxin.The present results suggest that fragment A of diphtheria toxin is capable of inhibiting the energy coupling in mitoplasts, thereby inhibiting protein synthesis. The detailed mechanism of the uncoupling and its possible physiological significance remains to be elucidated     

Mass characteristics of DNA obtained from chromosomes of a human carcinoma cell line

Sedimentation studies of DNA from chromosomes extracted from human mitotic cells showed that highmolecular-weight DNA can be obtained if cell hypotonic treatments and prolonged metaphase blocks are avoided. Two types of large double-stranded DNA were observed. One of these (M _r = 2.5×10⁸) appeared as a size class with characteristics reminiscent of the chromosomal DNA subunit hypothesis. However, this DNA is the decay product of larger molecules, whose minimum molecular weight is 6×10⁸.     

Estrogen regulation and ion dependence of taurine uptake by MCF-7 human breast cancer cells

It has been reported that estrogen receptor-positive MCF-7 cells express TauT, a Na⁺-dependent taurine transporter. However, there is a paucity of information relating to the characteristics of taurine transport in this human breast cancer cell line. Therefore, we have examined the characteristics and regulation of taurine uptake by MCF-7 cells. Taurine uptake by MCF-7 cells showed an absolute dependence upon extracellular Na⁺. Although taurine uptake was reduced in Cl^- free medium a significant portion of taurine uptake persisted in the presence of NO₃ ^-. Taurine uptake by MCF-7 cells was inhibited by extracellular β-alanine but not by L-alanine or L-leucine. 17β-estadiol increased taurine uptake by MCF-7 cells: the V_max of influx was increased without affecting the K_m. The effect of 17β-estradiol on taurine uptake by MCF-7 cells was dependent upon the presence of extracellular Na⁺. In contrast, 17β-estradiol had no significant effect on the kinetic parameters of taurine uptake by estrogen receptor-negative MDA-MB-231 cells. It appears that estrogen regulates taurine uptake by MCF-7 cells via TauT. In addition, Na⁺-dependent taurine uptake may not be strictly dependent upon extracellular Cl^-.     

Interference in DNA Replication Can Cause Mitotic Chromosomal Breakage Unassociated with Double-Strand Breaks

Morphological analysis of mitotic chromosomes is used to detect mutagenic chemical compounds and to estimate the dose of ionizing radiation to be administered. It has long been believed that chromosomal breaks are always associated with double-strand breaks (DSBs). We here provide compelling evidence against this canonical theory. We employed a genetic approach using two cell lines, chicken DT40 and human Nalm-6. We measured the number of chromosomal breaks induced by three replication-blocking agents (aphidicolin, 5-fluorouracil, and hydroxyurea) in DSB-repair-proficient wild-type cells and cells deficient in both homologous recombination and nonhomologous end-joining (the two major DSB-repair pathways). Exposure of cells to the three replication-blocking agents for at least two cell cycles resulted in comparable numbers of chromosomal breaks for RAD54^−/−/KU70^−/− DT40 clones and wild-type cells. Likewise, the numbers of chromosomal breaks induced in RAD54^−/−/LIG4^−/− Nalm-6 clones and wild-type cells were also comparable. These data indicate that the replication-blocking agents can cause chromosomal breaks unassociated with DSBs. In contrast with DSB-repair-deficient cells, chicken DT40 cells deficient in PIF1 or ATRIP, which molecules contribute to the completion of DNA replication, displayed higher numbers of mitotic chromosomal breaks induced by aphidicolin than did wild-type cells, suggesting that single-strand gaps left unreplicated may result in mitotic chromosomal breaks.     

Genetic analysis of the yeastCandida maltosa by means of induced parasexual processes

The usefulness of hybridization by protoplast fusion and mitotic segregation for the genetic analysis of the imperfect fodder yeastCandida maltosa was tested. Mitotically stable fusion hybrids were obtained with frequencies between 10^–6 and 10^–7. Complementation tests were performed by protoplast fusion. Substances that are known to induce frequent mitotic segregation in other yeast species such as benomyl, p-fluorophenylalanine, and acriflavine were ineffective inC. maltosa. UV irradiation induced mitotic segregation in up to 10%. This agent induced mainly mitotic crossing over inC. maltosa. Our data enabled the construction of the linkage group I with the sequenceCEN-ade-26-pro-1.     

The APC/C activator Cdh1 regulates the G2/M transition during differentiation of placental trophoblast stem cells

Differentiation of placental trophoblast stem (TS) cells to trophoblast giant (TG) cells is accompanied by transition from a mitotic cell cycle to an endocycle. Here, we report that Cdh1, a regulator of the anaphase-promoting complex/cyclosome (APC/C), negatively regulates mitotic entry upon the mitotic/endocycle transition. TS cells derived from homozygous Cdh1 gene-trapped (Cdh1^GT/GT) murine embryos accumulated mitotic cyclins and precociously entered mitosis after induction of TS cell differentiation, indicating that Cdh1 is required for the switch from mitosis to the endocycle. Furthermore, the Cdh1^GT/GT TS cells and placenta showed aberrant expression of placental differentiation markers. These data highlight an important role of Cdh1 in the G2/M transition during placental differentiation.     

DNA Synthesis and Fos and Jun Protein Expression in Mitotic and Postmitotic WI-38 Fibroblasts in Vitro

Normal human embryonic lung fibroblasts WI-38 differentiate spontaneously along the cell lineage mitotic fibroblasts (MF) I, II, and III and postmitotic fibroblasts (PMF) IV, V, VI, and VII in the fibroblast stem cell system in vitro, when appropriate methods are applied. The mitotic fibroblasts can be induced to shift to postmitotic fibroblasts by two treatments with mitomycin C (2× MMC) in a short period of time compared to spontaneous development. Mitotic and postmitotic fibroblast cell types have specific morphological and biochemical properties, e.g., [³⁵S]methionine polypeptide markers in 2D PAGE. Spontaneously arisen and experimentally induced (2× MMC) PMF have the same morphological and biochemical characteristics. Mitotic fibroblasts have 2n DNA and undergo DNA synthesis for reduplication. Postmitotic cells undergo, on average, two rounds of DNA synthesis for endoreduplication (polyploidization). Spontaneously arisen and experimentally induced postmitotic populations are composed of postmitotic fibroblasts PMF IV, V, and VI with 2n, 4n, and 8n DNA. DNA synthesis of mitotic and postmitotic WI-38 cell populations may be regulated by the expression of Fos and Jun proteins. The Fos level of MFs was higher by a factor of 15-24 and the Jun level of MFs by a factor of 4.2-6.3 than those of spontaneously arisen PMFs. In 2× MMC-induced PMFs, the Fos level was about 4.4-7.5 times higher and the Jun level 1.7-3.3 times higher than that of spontaneously arisen PMFs. The down-regulation of these two parameters is a normal event in the development of mitotic to postmitotic WI-38 fibroblasts in the fibroblast stem cell system and is not related to cellular aging.     

Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability ¹. Inaccuracies during chromosome separation are a hallmark of malignancy and associated with progressive disease ^2-4. The spindle assembly checkpoint (SAC) is a mitotic surveillance mechanism that holds back cells at metaphase until every single chromosome has established a stable bipolar attachment to the mitotic spindle¹. The SAC exerts its function by interference with the activating APC/C subunit Cdc20 to block proteolysis of securin and cyclin B and thus chromosome separation and mitotic exit. Improper attachment of chromosomes prevents silencing of SAC signaling and causes continued inhibition of APC/C^Cdc20 until the problem is solved to avoid chromosome missegregation, aneuploidy and malignant growths¹.Most studies that addressed the influence of improper chromosomal attachment on APC/C-dependent proteolysis took advantage of spindle disruption using depolymerizing or microtubule-stabilizing drugs to interfere with chromosomal attachment to microtubules. Since interference with microtubule kinetics can affect the transport and localization of critical regulators, these procedures bear a risk of inducing artificial effects ⁵.To study how the SAC interferes with APC/C-dependent proteolysis of cyclin B during mitosis in unperturbed cell populations, we established a histone H2-GFP-based system which allowed the simultaneous monitoring of metaphase alignment of mitotic chromosomes and proteolysis of cyclin B ⁶.To depict proteolytic profiles, we generated a chimeric cyclin B reporter molecule with a C-terminal SNAP moiety ⁶ (Figure 1). In a self-labeling reaction, the SNAP-moiety is able to form covalent bonds with alkylguanine-carriers (SNAP substrate) ^7,8 (Figure 1). SNAP substrate molecules are readily available and carry a broad spectrum of different fluorochromes. Chimeric cyclin B-SNAP molecules become labeled upon addition of the membrane-permeable SNAP substrate to the growth medium ⁷ (Figure 1). Following the labeling reaction, the cyclin B-SNAP fluorescence intensity drops in a pulse-chase reaction-like manner and fluorescence intensities reflect levels of cyclin B degradation ⁶ (Figure 1). Our system facilitates the monitoring of mitotic APC/C-dependent proteolysis in large numbers of cells (or several cell populations) in parallel. Thereby, the system may be a valuable tool to identify agents/small molecules that are able to interfere with proteolytic activity at the metaphase to anaphase transition. Moreover, as synthesis of cyclin B during mitosis has recently been suggested as an important mechanism in fostering a mitotic block in mice and humans by keeping cyclin B expression levels stable ^9,10, this system enabled us to analyze cyclin B proteolysis as one element of a balanced equilibrium ⁶.     

Temporal structure of S phase analyzed using an automated synchrony system

An automated system for cell cycle analysis is described in which synchronous populations of greater than 95% mitotic cells can be selected and subcultured without resorting to inhibitors or altered media. Selection of mitotic cells from roller bottle cultures generates synchronous populations of up to 5 × 10⁷ cells, as often as every 30 min, under conditions of constant temperature. A detailed analysis of the temporal pattern of DNA synthesis in the S phase of several Chinese hamster cells is presented. Several potential artefacts and uncertainties regarding the cell cycle blockage point in S phase synchronization are discussed.     

KCNMA1 Encoded Cardiac BK Channels Afford Protection against Ischemia-Reperfusion Injury

Mitochondrial potassium channels have been implicated in myocardial protection mediated through pre-/postconditioning. Compounds that open the Ca²⁺- and voltage-activated potassium channel of big-conductance (BK) have a pre-conditioning-like effect on survival of cardiomyocytes after ischemia/reperfusion injury. Recently, mitochondrial BK channels (mitoBKs) in cardiomyocytes were implicated as infarct-limiting factors that derive directly from the KCNMA1 gene encoding for canonical BKs usually present at the plasma membrane of cells. However, some studies challenged these cardio-protective roles of mitoBKs. Herein, we present electrophysiological evidence for paxilline- and NS11021-sensitive BK-mediated currents of 190 pS conductance in mitoplasts from wild-type but not BK^−/− cardiomyocytes. Transmission electron microscopy of BK^−/− ventricular muscles fibres showed normal ultra-structures and matrix dimension, but oxidative phosphorylation capacities at normoxia and upon re-oxygenation after anoxia were significantly attenuated in BK^−/− permeabilized cardiomyocytes. In the absence of BK, post-anoxic reactive oxygen species (ROS) production from cardiomyocyte mitochondria was elevated indicating that mitoBK fine-tune the oxidative state at hypoxia and re-oxygenation. Because ROS and the capacity of the myocardium for oxidative metabolism are important determinants of cellular survival, we tested BK^−/− hearts for their response in an ex-vivo model of ischemia/reperfusion (I/R) injury. Infarct areas, coronary flow and heart rates were not different between wild-type and BK^−/− hearts upon I/R injury in the absence of ischemic pre-conditioning (IP), but differed upon IP. While the area of infarction comprised 28±3% of the area at risk in wild-type, it was increased to 58±5% in BK^−/− hearts suggesting that BK mediates the beneficial effects of IP. These findings suggest that cardiac BK channels are important for proper oxidative energy supply of cardiomyocytes at normoxia and upon re-oxygenation after prolonged anoxia and that IP might indeed favor survival of the myocardium upon I/R injury in a BK-dependent mode stemming from both mitochondrial post-anoxic ROS modulation and non-mitochondrial localizations.     

能量化时线粒体内膜表面电荷的变化

本文报告用荧光探剂1,8—ANS和电泳激光光散射技术,研究鼠肝线粒体内膜在加入ATP的能量化过程中其膜表面电荷的变化。实验结果表明在加入ATP后线粒体内膜的能量化使其膜表面的负电荷减少。作者论讨了用上述二种方法研究线粒体内膜在能量化时表面电荷变化的有关问题。     

Gradual centriole maturation associates with the mitotic surveillance pathway in mouse development

Centrosomes, composed of two centrioles and pericentriolar material, organize mitotic spindles during cell division and template cilia during interphase. The first few divisions during mouse development occur without centrioles, which form around embryonic day (E) 3. However, disruption of centriole biogenesis in Sas‐4 null mice leads to embryonic arrest around E9. Centriole loss in Sas‐4 ^−/− embryos causes prolonged mitosis and p53‐dependent cell death. Studies in vitro discovered a similar USP28‐, 53BP1‐, and p53‐dependent mitotic surveillance pathway that leads to cell cycle arrest. In this study, we show that an analogous pathway is conserved in vivo where 53BP1 and USP28 are upstream of p53 in Sas‐4 ^−/− embryos. The data indicate that the pathway is established around E7 of development, four days after the centrioles appear. Our data suggest that the newly formed centrioles gradually mature to participate in mitosis and cilia formation around the beginning of gastrulation, coinciding with the activation of mitotic surveillance pathway upon centriole loss.     

Mitotic segregation in hybrid of methylotrophic yeastCandida pelliculosa

Protoplasts from two autoxotrophic mutants of methylotrophic yeastCandida pelliculosa were fused by the PEG technique, and the genetic structure of the hybrid obtained was studied by means of mitotic segregation. Spontaneous mitotic segregation depended on the composition of the medium and the type of carbon source used. Mechanisms by which segregants appeared were studied by means of UV- and benomyl-induced mitotic segregation. Our data suggest that one large percentage of segregants (Leu ^–) occurred as a result of mitotic chromosome loss. The other large portion of segregants (Ade ^– andLys ^–) are a consequence of mitotic recombination.     

CDK-dependent phosphorylation of Alp7–Alp14 (TACC–TOG) promotes its nuclear accumulation and spindle microtubule assembly

As cells transition from interphase to mitosis, the microtubule cytoskeleton is reorganized to form the mitotic spindle. In the closed mitosis of fission yeast, a microtubule-associated protein complex, Alp7–Alp14 (transforming acidic coiled-coil–tumor overexpressed gene), enters the nucleus upon mitotic entry and promotes spindle formation. However, how the complex is controlled to accumulate in the nucleus only during mitosis remains elusive. Here we demonstrate that Alp7–Alp14 is excluded from the nucleus during interphase using the nuclear export signal in Alp14 but is accumulated in the nucleus during mitosis through phosphorylation of Alp7 by the cyclin-dependent kinase (CDK). Five phosphorylation sites reside around the nuclear localization signal of Alp7, and the phosphodeficient alp7-5A mutant fails to accumulate in the nucleus during mitosis and exhibits partial spindle defects. Thus our results reveal one way that CDK regulates spindle assembly at mitotic entry: CDK phosphorylates the Alp7–Alp14 complex to localize it to the nucleus.     

1H, 13C and 15N resonance assignments of the kinetochore localisation domain of BUBR1, a central component of the spindle assembly checkpoint

Human BUBR1 is a 120 kDa protein that plays a central role in the spindle assembly checkpoint (SAC), the evolutionary conserved and self-regulatory system of higher organisms that monitors and repairs defects in chromosome segregation in mitotic cells. BUBR1 is organised into several domains, with an N-terminal region responsible for its localisation into the kinetochore, the multi-component proteinaceous network that assembles onto chromosomes upon mitotic entry. We have expressed and purified uniformly-¹⁵N/¹³C N-terminal BUBR1 and assigned backbone and side-chain resonances bound to an unlabelled peptide from the protein Blinkin, an element essential for recruitment of BUBR1 to the kinetochore. These assignments provide insights on the Blinkin interaction interface and form the basis of the three-dimensional structure determination of a BUBR1-Blinkin complex.