Gaspar Banfalvi

Cell cycle synchronization of animal cells and nuclei by centrifugal elutriation

Synchronization of cells and nuclei is a powerful technique for the exact study of regulatory mechanisms and for understanding cell cycle events. Counterflow centrifugal elutriation is a biophysical cell separation technique in which cell size and sedimentation density differences of living cells are exploited to isolate subpopulations in various stages of cell cycle. Here, a protocol is described for the separation of phase-enriched subpopulations from exponentially growing Chinese hamster ovary cells at high-resolution power of elutriation. The efficiency of elutriation is confirmed by measuring the DNA content fluorimetrically and by flow cytometry. The resolution power of elutriation is demonstrated by the ability to fractionate nuclei of murine pre-B cells. The installation and elutriation by collecting 16–30 synchronized fractions, including particle size analysis, can be achieved in 4–5 h.

Comparative study of cyanotoxins affecting cytoskeletal and chromatin structures in CHO-K1 cells

In this study we compared the effects of the two frequently occuring and most dangerous cyanobacterial toxins on the cellular organization of microfilaments, microtubules and on the chromatin structure in Chinese hamster ovary (CHO-K1) cells. These compounds are the widely known microcystin-LR (MC-LR) and cylindrospermopsin (CYN) classified as the highest-priority cyanotoxin. Toxic effects were tested in a concentration and time dependent manner. The hepatotoxic MC-LR did not cause significant cytotoxicity on CHO-K1 cells under 20 microM, but caused apoptotic changes at higher concentrations. Apoptotic shrinkage was associated with the shortening and loss of actin filaments and with a concentration dependent depolymerization of microtubules. No necrosis was observed over the concentration range (1-50 microM MC-LR) tested. Cylindrospermopsin did cause apoptosis at low concentrations (1-2 microM) and over short exposure periods (12h). Necrosis was observed at higher concentrations (5-10 microM) and following longer exposure periods (24 or 48h). Cyanotoxins also affected the chromatin structure. The condensation process was inhibited by MC-LR at a later stage and manifested as broken elongated prechromosomes. CYN inhibited chromatin condensation at the early fibrillary stage leading to blurred fluorescent images of apoptotic bodies and preventing the formation of metaphase chromosomes. Cylindrospermopsin exhibited a more pronounced toxic effect causing cytoskeletal and nuclear changes as well as apoptotic and necrotic alterations.

Cellular Effects of Heavy Metals

Introduction. Chapter 1. Heavy Metals, Trace Elements and their Cellular Effects. I. Heavy metal toxicity in microbes. Chapter 2. Toxic Metal/Metalloid Tolerance in Fungi - A Biotechnology-Oriented Approach. Chapter 3. Interference of chromium with cellular functions. Chapter 4. Saccharomyces cerevisiae as a Model Organism for Elucidating Arsenic Tolerance Mechanisms. II. Heavy metal induced toxicity in insect cells. Chapter 5 . Heavy Metal Toxicity in an Insect Cell Line (Methyl-HgCl, HgCl2, CdCl2 and CuSO4) . III. Genotoxic effects of heavy metals. Chapter 6. Cellular Changes in Mammalian Cells Induced by Cadmium. Chapter 7. Chromatin toxicity of Ni(II) Ions in K562 Erythroleukemia Cells. Chapter 8. Genotoxic Chromatin Changes in Schizosaccharomyces pombe Induced by Hhexavalent Chromium (CrVI) Ions. Chapter 9. Chromatin Changes upon Silver Nitrate Treatment in Human Keratinocyte HaCaT and K562 Erythroleukemia Cells. IV. Chemical carcinogenesis induced by heavy metals. Chapter 10. Heavy Metal-Induced Carcinogenicity: Depleted Uranium and Heavy Metal. Chapter 11. Role of Oxidative Damage in Metal-Induced Carcinogenesis. V. Cellular responses to heavy metal exposure. Chapter 12. Non-native Proteins as Newly-identified Targets of Heavy Metals and Metalloids. Chapter 13. Cellular Mechanisms to Respond to Cadmium Exposure: Ubiquitin Ligases. Chapter 14. Metals Induced Disruption of Ubiquitin Proteasome System, Activation of Stress Signaling and Apoptosis. VI. Biomakers. Chapter 15. Blood Lead Level (BLL, B-Pb) in Human and Animal Populations: B-Pb as a Biological Marker to Environmental Lead Exposure. VII. Removal of heavy metals. Chapter 16. Removal of Heavy Metal Sulfides and Toxic Contaminants from Water.

Changes in the expression and distribution of the inducible and endothelial nitric oxide synthase in mucosal biopsy specimens of inflammatory bowel disease

Objective The role of nitric oxide (NO) in the pathophysiology of inflammatory bowel disease (IBD) is controversial. The aim of this study was to investigate the expression and localization of nitric oxide synthase isoforms (iNOS, eNOS) in IBD colonic mucosa. Material and methods Forty-four patients with IBD (24 ulcerative colitis (UC), 20 Crohns disease (CD) and 16 controls) were investigated by colonoscopy. iNOS and eNOS in tissue sections was demonstrated by histochemistry (NADPH-diaphorase reaction) and immunohistochemistry. Cell type analysis and quantitative assessment of the iNOS immunoreactive (IR) cells and densitometry of iNOS in immunoblots were also performed. Results iNOS-IR cells were significantly numerous in inflamed mucosa of UC (64±4 cells/mm2) than in CD (4±2 cells/mm2). iNOS-IR/CD15-IR cells showed significant elevation in inflamed (i) versus uninflamed (u) UC mucosa (UCu 8±3%, UCi 85±10%) In CD, the percentage of iNOS-IR/CD68-IR cells was lower in inflamed sites (CDu 23±8%, CDi 4±3%). Immunoblot of biopsies revealed significant elevation of iNOS in active UC compared with uninflamed sites, whereas in CD no significant changes were detected. Differences were observed in eNOS and endothelial marker CD31 immunoreactivity. In patients with UC and in controls the ratios of eNOS/CD31-IR vessels were 82.3% and 92.0% respectively, whereas in CD the ratio was 8.3% with a concomitantly significant increase of CD31-IR vessels. The distribution and morphological characteristics of the NOS-IR inflammatory cells and endothelia were similar to those showing NADPH-diaphorase reactivity. Conclusions Differences observed in the expression and distribution of NOS isoforms in immune and endothelial cells may contribute to better understanding of the structural and physiological changes in UC and CD.

Cadmium induced apoptotic changes in chromatin structure and subphases of nuclear growth during the cell cycle in CHO cells

CHO cells were grown in the presence of 1 μ M CdCl2 and subjected to ATP-dependent replicative DNA synthesis after permeabilization. By decreasing the density of the cell culture replicative DNA synthesis was diminishing. At higher than 2 × 106 cell/ml concentration Cd had virtually no effect on the rate of DNA replication. Growth at higher cell concentrations could be supressed by increasing Cd concentration. After Cd treatment cells were synchronized by counterflow centrifugal elutriation. Cadmium toxicity on cell growth in early and mid S phase led to the accumulation of enlarged cells in late S phase. Flow cytometry showed increased cellular and nuclear sizes after Cd treatment. As the cells progressed through the S phase, 11 subpopulations of nuclear sizes were distinguished. Apoptotic chromatin changes were visualized by fluorescent microscopy in a cell cycle dependent manner. In the control untreated cells the main transitory forms of chromatin corresponded to those we have published earlier (veil-like, supercoiled chromatin, fibrous, ribboned structures, chromatin strings, elongated prechromosomes, precondensed chromosomes). Cadmium treatment caused: (a) the absence of decondensed veil-like structures and premature chromatin condensation in the form of apoptotic bodies in early S phase (2.2–2.4 average C-value), (b) the absence of fibrous structures, the lack of supercoiled chromatin, the appearance of uncoiled ribboned chromatin and perichromatin semicircles, in early mid S phase (2.5–2.9 C), (c) the presence of perichromatin fibrils and chromatin bodies in mid S phase (2.9–3.2 C), (d) early intra-nuclear inclusions, elongated forms of premature chromosomes, the extrusion and rupture of nuclear membrane later in mid S phase (3.3–3.4 C), (e) the exclusion of chromatin bodies and the formation of clusters of large-sized perichromatin granules in late S phase (3.5–3.8 C) and (f) large extensive disruptions and holes in the nuclear membrane and the clumping of incompletely folded chromosomes (3.8–4. C).

Overview of Cell Synchronization

Widespread interest in cell synchronization is maintained by the studies of control mechanisms involved in cell cycle regulation. During the synchronization distinct subpopulations of cells are obtained representing different stages of the cell cycle. These subpopulations are then used to study regulatory mechanisms of the cycle at the level of macromolecular biosynthesis (DNA synthesis, gene expression, protein synthesis), protein phosphorylation, development of new drugs, etc. Although several synchronization methods have been described, it is of general interest that scientists get a compilation and an updated view of these synchronization techniques. This introductory chapter summarizes: (1) the basic concepts and principal criteria of cell cycle synchronizations, (2) the most frequently used synchronization methods, such as physical fractionation (flow cytometry, dielectrophoresis, cytofluorometric purification), chemical blockade, (3) synchronization of embryonic cells, (4) synchronization at low temperature, (5) comparison of cell synchrony techniques, (6) synchronization of unicellular organisms, and (7) the effect of synchronization on transfection.

A possible stimulatory effect of FMRFamide on neural nitric oxide production in the central nervous system of Helix lucorum L.

The anatomical and functional relationship between neurons expressing nitric oxide (NO) synthase and molluscan cardioexcitatory (FMRFamide)-like neuropeptides was studied in the central ganglia of Helix lucorum (Pulmonata, Gastropoda), applying NADPHdiaphorase (NADPHd) histochemistry to visualize NO synthase and immunocytochemistry to demonstrate FMRFamide (FMRFa) at the light microscopic level. The NO production of the ganglia was detected by the colorimetric Griess determination of nitrite, a breakdown product of NO. Effects of the NO synthase substrate amino acid L-arginine, the NO synthase inhibitor Nω-nitro-L-arginine (NOARG), synthetic FMRFa and the FMRFa sensitive ion channel blocker amiloride hydrochloride on nitrite production were also tested. NADPHd reaction labeled nerve cells and fibers in the procerebra, mesocerebra and metacerebra within the cerebral ganglia, and cell clusters in the postcerebral ganglia. FMRFa immunolabeling could be observed within subpopulations of NADPHd positive cells and in pericellular varicose fibers surrounding NADPHd stained neurons. Nitrite production of the ganglia was stimulated by L-arginine (10– 20 mM) but was decreased by NOARG (1–2 mM). Synthetic FMRFa (0.830–3.340 mM) increased the nitrite production in a dose dependent manner, but was ineffective in the presence of NOARG. Amiloride hydrochloride (7.890 mM) reduced the FMRFa evoked nitrite production in all ganglia. This is the first description of an anatomical relationship between putative NO producing and FMRFa containing cells, suggesting a possible regulatory role of FMRFa in the NO mediated signaling in an invertebrate nervous system.

Gamma irradiation-induced apoptosis in murine pre-B cells prevents the condensation of fibrillar chromatin in early S phase

Local changes in chromatin structure leading to temporally distinct geometric forms were characterized in nuclei of reversibly permeabilized cells. Reversal of permeabilization was tested by 3H-thymidine incorporation and trypan blue dye exclusion. Apoptotic changes were visualized in a cell cycle dependent manner at the chromatin level by fluorescent microscopy in non-irradiated cells and after 400 rad Co60 irradiation. Fluorescent microscopy of chromatin structures belonging mainly to the interphase of the cell cycle confirmed the existence of specific geometric forms in nuclei of non-irradiated cells. In this control population, the following main transitory forms of condensing chromatin were distinguished: decondensed veil-like structures and fibrous structures in early and mid S phase (2.0–2.5 average C-value), chromatin bodies, semicircles later in mid S phase (3.0–3.5 C), precondensed chromosomes in late S (3.5–3.7 C) and metaphase chromosomes at the end and after S phase (3.7–4.0 C). Our results show that upon γ-irradiation (a) the cellular and nuclear sizes were increased, (b) the DNA content was lower in each elutriated subpopulation of cells, (c) the progression of the cell cycle was arrested in the early S phase at 2.4 C value, (d) the chromatin condensation was blocked between the fibrillar chromatin and precondensed elongated chromosomal forms, and (e) the number and size of apoptotic bodies were inversely correlated with the progression of the cell cycle, with many small apoptotic bodies in early S phase and less and larger apoptotic bodies in late S phase.

Time-lapse analysis of cell death in mammalian and fungal cells.

Time-lapse video microscopy was designed to follow the movement of single cells for an unlimited period of time under physiological conditions. The system is based on two inverted microscopes located in a CO(2) incubator and equipped with charge-coupled device cameras connected to the computer. Frames were recorded every minute and the subsequent video sequence was converted to database form. The system was applied to describe the movements of normal HaCaT cells and Pb-treated cells causing the so-called apoptotic dance during cell death. The apoptotic movement was also followed in high-osmolarity glycerol-type mitogen-activated protein kinase (MAPK) null mutant of Fusarium proliferatum, a filamentous fungus, during osmotic stress. The shortest (20 min) and most vigorous death movements were observed in apoptotic fungal cells subjected to salt stress. The necrotic process at higher Pb concentration (50 microM) took 2-3 h, whereas the apoptotic process at lower Pb concentrations lasted from minutes to days.

Heavy Metals, Trace Elements and Their Cellular Effects

The book starts with the brief review of chapters. In this chapter heavy metals have been redefined as those trace elements that have ≥ 3 g/cm3 densities and may cause harmful biological effects. The chapter arrived to this definition by clarifying first the light elements on the basis of their electronic configurations and compatibility with those of bioelements (CHNOPS group) in constructing biomolecules. As compatibility criteria the chemical bond formation between s–p electrons and p–p electrons were taken, allowing the tetrahedral three dimensional construction of biological compounds with four bonding partners. The compatibility range ended at 1s22s22p63s23p64s2 electronic configuration corresponding to calcium, which is the 20th element in the periodic table. From element 21 (Sc) the wide range of redox behavior, high reactivity, rich coordination chemistry and complex formation of transition metals is due to the outer d and f electron subshells and explain their important catalytic role in enzyme reactions and toxicity at higher cellular concentrations. The chapter describes the most important cellular effects of heavy metals. The advantages of changing from in vivo to in vitro cellular systems have been pointed out. The methods for the detection and determination of heavy metals in cells are summarized.