Similar replicon properties of higher plant cells with different S periods and genome sizes

Root meristematic cells of nine unrelated diploid higher plants with genome sizes that differ 82-fold and with S periods that differ 4-fold have similar replicon sizes and single replication fork rates that average 22 μm and 8 μm/h respectively. The average replicon size of 22 μm is near the 18 μm obtained by extrapolation of measurements, taken from DNA fiber autoradiograms, to zero pulse time with [³H]thymidine. The data suggest that the duration of S is determined by the minimal number of replicon families that function sequentially during DNA replication.     

In vivo action of valinomycin on Ehrlich ascites tumor cells. Inhibition of the proliferative activity of the tumor cells under the action of valinomycin]

It was shown that repeated administrations of valinomycin in doses of 1.0 and 0.01 gamma/gm to mice with Ehrlich ascitic tumors inhibited the ascite development. The radioautographic study using 3H-thimidine showed that 24 hours after the administration of valinomycin in doses of 0.1 and 0.01 gamma/gm transference of the cells into the phase of DNA synthesis was inhibited--inhibition of the tumor cell mitotic activity took place.     

Respiratory repression and the stability of the mitochondrial genome

Summary The variation in sensitivity of the mitochondrial genome of Saccharomyces cerevisiae to ethidium bromide-induced petite mutation in response to changes in glucose concentration has been studied. Growth in high glucose considerably depressed the mutation rate, whilst small variations are observed in response to step-up or step-down in glucose concentration. Variations in mitochondrial DNA and respiratory activity during the mutagenic process are described. Effects of non-metabolizable sugars which repress mitochondrial biogenesis and a number of antimitochondrial drugs have been investigated. The results are discussed in terms of possible mechanisms of modulation of the mutation rates.     

Cytofluorometric DNA base determination in vertebrate species with different genome sizes

The base composition of DNA was studied in 15 amphibian species and 28 reptile species by means of DAPI, a fluorochrome specific for adenine-thymine rich DNA (AT-rich DNA). The results obtained in reptiles and anuran amphibians coincided with biochemical data available for some species. In urodeles, on the contrary, the findings contrasted with biochemical data and suggest that DAPI is unable to stain all the AT-rich DNA in the erythrocytes of these organisms. It is concluded that the method is suitable for studying species with a small genome size, such as reptiles and anuran amphibians, but is not suitable for nuclei with a large genome size and a highly compact chromatin, such as urodele erythrocytes.     

Characterization of cells with different mitochondrial membrane potential during apoptosis.

BACKGROUND: Until now, the simultaneous analysis of several parameters during apoptosis, including DNA content and mitochondrial membrane potential (DeltaPsi), has not been possible because of the spectral characteristics of the commonly used dyes. Using polychromatic flow cytometry based upon multiple laser and UV lamp excitation, we have characterized cells with different DeltaPsi during apoptosis. METHODS: U937 cells were treated with the flavonoid quercetin (Qu) and stained with JC-1 to detect DeltaPsi, propidium iodide (PI) for cell viability, Hoechst 33342 for DNA content, Annexin V conjugated with Alexa Fluor-647 for detection of phosphatidilserine (PS) exposure, marker of early apoptosis, or Mitotracker Deep Red for the determination of mitochondrial mass. RESULTS: Treatment with Qu provoked the onset of three cell populations with different DeltaPsi: (1) healthy cells, with normal DeltaPsi, DNA content and physical parameters, high mitochondrial mass, PI- and Annexin V-negative; (2) cells with intermediate DeltaPsi and normal DNA content, but with physical parameters typical of apoptotic cells and low mitochondrial mass; most of them were PI+ and Annexin V+; (3) cells with collapsed DeltaPsi that had low mitochondrial mass and were Annexin-V+, PI+; half of them showed diminished DNA content. Similar results, i.e. the presence of cells with intermediate DeltaPsi, were observed in other models of apoptosis. CONCLUSIONS: During Qu-induced apoptosis, loss of DeltaPsi, PS exposure, and decrease of mitochondrial mass are early events that precede permeability to PI and loss of DNA. Populations of cells with different DeltaPsi, as revealed by flow cytometry after JC-1 staining, differed also for other parameters associated to apoptosis. Thus, the simultaneous analysis of several parameters by polychromatic flow cytometry permits a better identification of many stages of cell death, and, more in general, allows to evaluate the eventual heterogenic sensibility of the population under study to a given compound.     

Capsaicin induces cytotoxicity in pancreatic neuroendocrine tumor cells via mitochondrial action

Capsaicin (CAP), the pungent ingredient of chili peppers, inhibits growth of various solid cancers via TRPV1 as well as TRPV1-independent mechanisms. Recently, we showed that TRPV1 regulates intracellular calcium level and chromogranin A secretion in pancreatic neuroendocrine tumor (NET) cells. In the present study, we characterize the role of the TRPV1 agonist – CAP – in controlling proliferation and apoptosis of pancreatic BON and QGP-1 NET cells. We demonstrate that CAP reduces viability and proliferation, and stimulates apoptotic death of NET cells. CAP causes mitochondrial membrane potential loss, inhibits ATP synthesis and reduces mitochondrial Bcl-2 protein production. In addition, CAP increases cytochrome c and cleaved caspase 3 levels in cytoplasm. CAP reduces reactive oxygen species (ROS) generation. The antioxidant N-acetyl-l-cysteine (NAC) acts synergistically with CAP to reduce ROS generation, without affecting CAP-induced toxicity. TRPV1 protein reduction by 75% reduction fails to attenuate CAP-induced cytotoxicity. In summary, these results suggest that CAP induces cytotoxicity by disturbing mitochondrial potential, and inhibits ATP synthesis in NET cells. Stimulation of ROS generation by CAP appears to be a secondary effect, not related to CAP-induced cytotoxicity. These results justify further evaluation of CAP in modulating pancreatic NETs in vivo.     

Expression of the mitochondrial genome in HeLa cells. XVI. Electrophoretic properties of the products of in vivo and in vitro mitochondrial protein synthesis

The size distribution of the proteins synthesized by isolated HeLa cell mitochondria has been analyzed by polyacrylamide gel electrophoresis and compared to that of the in vivo products of mitochondrial protein synthesis.The electrophoretic pattern of the mitochondrial proteins labeled in vitro with [³H]leucine has a group of partially resolved components migrating in the region corresponding to 12,000 to 25,000 molecular weight, and another group, more abundant, in the range from 40,000 to 55,000 molecular weight. This pattern is very similar, after a two-hour incubation of mitochondria, to that of the proteins labeled in vivo in a 30-minute [³H]leucine pulse.     

Cytotoxic action of polymorphonuclear leukocytes on tumor and normal cells during ascite tumor development in vitro and in vivo

A vast number of studies, including the authors' own research, support the important role polymorphonuclear leukocytes (PMNL) in the development of ascite tumors. The method of luminol-dependent chemiluminescence (CL) was used to show the presence of two functionally different PMNL pools in a tumor-bearing organism: 1) "primed" PMNL, which circulate in the blood stream, and 2) "activated" PMNL, which are accumulated in the tumor zone and are capable of spontaneous CL. The purpose of the present investigation was to compare cytotoxic effects of primed and activated PMNL on tumor cells (ascite Ehrlich carcinoma (AEC), ascite Zajdel hepatoma) upon co-cultivation, as well as on normal cells of the organism, erythrocytes in vitro and in vivo. Upon stimulation with phorbol myristate acetate (PMA), PMNL effectively damaged AEC cells within the first 24 h until PMNL apoptosis occurred. Upon further co-cultivation, the tumor cells grew in number, which suggest the participation of PMNL in tumor protection. When stimulated with PMNL, pools suppressed tumor growth in vitro, since in this case the cytotoxicity was due to both reactive oxygen species and proteolytic enzymes. As it has been shown earlier by the authors, the functional potential of PMNL increases many times during tumor growth, and we suggested that not only tumor but also normal cells could be damaged. In this connection, we have studied the cytotoxic effect of primed and activated PMNL on rat erythrocytes in vitro on their co-cultivation. On stimulation with PMA, the rate of lysis of erythrocytes by primed PMNL increase many times compared to the norm. The fMLP-stimulated cytotoxity was 1.5-2.0 times higher than in the norm. Activated PMNL without stimulation are capable of producing only a partial lysis of erythrocytes (5-7 %). In order to assess the cytotoxic action of PMNL on erythrocytes in vivo, the hemoglobin content in erythrocytes and blood plasm of rats was measured in the course of tumor growth. The hemoglobin content in erythocytes during growth tumor decreased from 135 +/- 10 to 85 +/- 5 g/l, whereas in the blood plasm the hemoglobin content gradually increased by almost two times. The results enable us to suggest that one of death causes of tumor-bearing organisms may be the cytotoxic action of PMNL on normal cells of the organism caused by hyperproduction of ROS.     

Growth of eukaryotic cells in relation to the structure of mitochondrial membranes and mitochondrial genome

Viability ofpetite-negative yeast, such asKluyveromyces lactis, is dependent on functional mitochondrial genome encoding essential components of both mitochondrial protein synthesizing system and oxidative phosphorylation. We have isolated several nuclear mutants impaired in mitochondrial functions that were unable to grow on non-fermentable carbon and energy sources. They were used for the isolation and molecular characterization of the three genes encoding apocytochromec, apocytochromec ₁ and the protein involved in the biogenesis of cytochrome oxidase. All cytochrome-deficient mutants were viable and did not survive the ethidium bromide mutagenesis.Petite-positiveSaccharomyces cerevisiae requires intact mitochondrial genome when its phosphatidylglycerolphosphate synthase was inactivated due to mutation in thePEL1 gene. UsingPEL-lacZ fusion genes it was demonstrated that Pel1p is a mitochondrial protein (expressed in response tomyo-inositol and choline). Thepel1 mutant was deficient in phosphatidylglycerol (PG) and cardiolipin (CL) and itsrho ⁻/rho ⁰ mutants grew extremely slowly on complex medium with glucose. Under the same conditions the growth rate of thecrd1 rho ⁻ double mutants was similar to that of its parentcrd1 mutant deficient in cardiolipin synthase and accumulating PG. The results demonstrate that thepetite negativity in yeast is not dependent on an intact respiratory chain or functional oxidative phosphorylation. The presence of the negatively charged PG or CL seems to be essential for the maintenance of specific mitochondrial functions required for the normal mitotic growth of yeast cells. Presented at theInternational Conference on Recent Problems in Microbiology and Immunology, Košice (Slovakia), 13–15 October 1999.     

Use of spectroscopic and zeta potential techniques to study the interaction between lysozyme and curcumin in the presence of silver nanoparticles at different sizes

This article describes, for the first time, the effect of three different sizes of silver nanoparticles on the binding of curcumin to lysozyme as examined by spectroscopic and zeta potential techniques at physiological conditions. The binding constants of curcumin to lysozyme in the presence of silver nanoparticles were measured. Based on the results of synchronous fluorescence and three-dimensional fluorescence spectroscopy, the presence of the different sizes of silver nanoparticles caused conformational changes in lysozyme during the binding of curcumin. Such changes were also observed when increasing the curcumin concentration. The results of fluorescence resonance energy transfer theory indicated that different sizes of silver nanoparticles could change the binding distance between curcumin and lysozyme. Based on the red edge excitation shift approach, we concluded that the limited mobility around the Trp residues decreased in the presence of silver nanoparticles with bigger size. Under resonance light scattering, the aggregation of curcumin on lysozyme in the presence of silver nanoparticles can play a major role in functional proteins.

Communicated by Ramaswamy H. Sarma     

Multiparametric analysis of cells with different mitochondrial membrane potential during apoptosis by polychromatic flow cytometry

The analysis of changes in mitochondrial membrane potential (MMP) that can occur during apoptosis provides precious information on the mechanisms and pathways of cell death. For many years, the metachromatic fluorochrome JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide) was used for this purpose. Thanks to new dyes and to the technical improvements recently adopted in several flow cytometers, it is now possible to investigate, along with MMP, a variety of other parameters. Using three sources of excitation and polychromatic flow cytometry, we have developed a protocol that can be applied to cells undergoing apoptosis. In the model of U937 cells incubated with the chemopreventive agent quercetin (3,3',4',5,7-pentahydroxyflavone), we describe the detection at the single cell level of changes in MMP (by JC-1), early apoptosis (exposition of phosphatidylserine on the plasma membrane detected by annexin-V), late apoptosis and secondary necrosis (decreased DNA content by Hoechst 33342 and permeability of the plasma membrane to propidium iodide). The procedure can be completed in less than 2 h.     

Length and interspersion of repetitive and non repetitive DNA sequences in four Amphibian species with different genome sizes

The interspersion period of repetitive and unique sequences was analyzed by two different methods, electron microscopy and agarose gel electrophoresis, for four Amphibian species with different nuclear DNA content, namely the Anura Xenopus laevis (3 pg DNA per haploid genome) and Bufo bufo (7 pg) and the Urodela Triturus cristatus (23 pg) and Necturus maculosus (52 pg). Within each of the two subclasses it has been found that interspecific differences, in DNA content, due to variations in the amount of repetitive sequences, do not involve variations in length of the interspersed repetitive sequences. They remain about 380 base pairs. Furthermore, the unique sequences length has been found to be shorter in Bufo (760 base pairs) than in Xenopus (1600) and in Necturus (880) than in Triturus (1340). A study of the interspersion period has shown that the great difference in DNA content between Anura and Urodela, which had been previously shown not to have involved changes in the relative amounts of the various sequence classes, does not involve changes in the interspersion period.     

The roles of fission yeast exonuclease 5 in nuclear and mitochondrial genome stability

The Exo5 family consists of bi-directional, single-stranded DNA-specific exonucleases that contain an iron-sulfur cluster as a structural motif and have multiple roles in DNA metabolism. S. cerevisiae Exo5 is essential for mitochondrial genome maintenance, while the human ortholog is important for nuclear genome stability and DNA repair. Here, we identify the Exo5 ortholog in Schizosaccharomyes pombe (spExo5). The activity of spExo5 is highly similar to that of the human enzyme. When the single-stranded DNA is coated with single-stranded DNA binding protein RPA, spExo5 become a 5′-specific exonuclease. Exo5Δ mutants are sensitive to various DNA damaging agents, particularly interstrand crosslinking agents. An epistasis analysis places exo5⁺ in the Fanconi pathway for interstrand crosslink repair. Exo5⁺ is in a redundant pathway with rad2⁺, which encodes the flap endonuclease FEN1, for mitochondrial genome maintenance. Deletion of both genes lead to severe depletion of the mitochondrial genome, and defects in respiration, indicating that either spExo5 or spFEN1 is necessary for mitochondrial DNA metabolism.     

Measurement of 'in situ' mitochondrial membrane potential in Ehrlich ascites tumor cells during aerobic glycolysis

(1) A method is presented for continuous and simultaneous monitoring of the 'in situ' mitochondrial membrane potential (delta psi m) and respiration rate of Ehrlich ascites tumor cells. The method involves permeabilization of the plasma membrane, achieved by treatment with low digitonin concentration, and the use of a TPP+ selective electrode attached to an oxygraph vessel. Binding of the probe inside the cells was analyzed assuming a proportional relationship between the amount of bound TPP+ and the free concentration of the lipophilic cation. (2) Evidence is reported that the addition of glucose to digitonin-permeabilized Ehrlich ascites tumor cells causes a decrease of mitochondrial membrane potential that coincided with a transient enhancement of the respiration rate and remained unchanged during the subsequent Crabtree effect. We have characterized the effect of glucose on delta psi m by determining its dependent on the glycolytic pathway and its sensitivity towards oligomycin. The mutual relationships between glucose and ADP effects on the mitochondrial membrane potential were also studied. A plausible mechanism underlying the depolarization of mitochondrial membrane induced by glucose is presented.