Cellular growth and the preparation of cells for division between two successive cell divisions is called the cell cycle. The cell cycle is divided into several phases; the length of these particular cell cycle phases is an important characteristic of cell life. The progression of cells through these phases is a highly orchestrated process governed by endogenous and exogenous factors. For the elucidation of the role of these factors, including pathological aspects, various methods have been developed. Among these methods, those focused on the analysis of the duration of distinct cell cycle phases play important role. The main aim of this review is to guide the readers through the basic methods of the determination of cell cycle phases and estimation of their length, with a focus on the effectiveness and reproducibility of the described methods.
A synchronous population of cells is one of the prerequisites for studying cell cycle processes such as DNA replication, nuclear and cellular division. Green algae dividing by multiple fission represent a unique single cell system enabling the preparation of highly synchronous cultures by application of a light-dark regime similar to what they experience in nature. This chapter provides detailed protocols for synchronization of different algal species by alternating light-dark cycles; all critical points are discussed extensively. Moreover, detailed information on basic analysis of cell cycle progression in such cultures is presented, including analyses of nuclear, cellular, and chloroplast divisions. Modifications of basic protocols that enable changes in cell cycle progression are also suggested so that nuclear or chloroplast divisions can be followed separately.
- MeSH
- Staining and Labeling methods MeSH
- Cell Division MeSH
- Cell Culture Techniques methods MeSH
- Cell Cycle MeSH
- Chlamydomonas reinhardtii cytology genetics growth & development MeSH
- Chlorophyta cytology genetics growth & development MeSH
- Chloroplasts genetics MeSH
- DNA, Plant genetics MeSH
- Photoperiod * MeSH
- Cell Fractionation methods MeSH
- DNA Replication MeSH
- Light MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
A comparative two-photon excitation spectroscopic study of the exciton structure of the core antenna complex (LH1) and its subunit B820 was carried out. LH1 and its subunit B820 were isolated from cells of the carotenoid-less mutant G9 of Rhodospirillum rubrum. The measurements were performed by two-photon pump-probe spectroscopy. Samples were excited by 70 fs pulses at 1390 nm at a frequency of 1 kHz. Photoinduced absorption changes were recorded in the spectral range from 780 to 1020 nm for time delays of the probe pulse relative to the pump pulse in the - 1.5 to 11 ps range. All measurements were performed at room temperature. Two-photon excitation caused bleaching of exciton bands (k = 0, k = ± 1) of the circular bacteriochlorophyll aggregate of LH1. In the case of the B820 subunit, two-photon excitation did not cause absorption changes in this spectral range. It is proposed that in LH1 upper exciton branch states are mixed with charge-transfer (CT) states. In B820 such mixing is absent, precluding two-photon excitation in this spectral region. Usually, CT states are optically "dark", i.e., one photon-excitation forbidden. Thus, their investigation is rather complicated by conventional spectroscopic methods. Thus, our study provides a novel approach to investigate CT states and their interaction(s) with other excited states in photosynthetic light-harvesting complexes and other molecular aggregates.
Cell quantification is widely used in basic or applied research. The current sensitive methods of cell quantification are exclusively based on the analysis of non-fixed cells and do not allow the simultaneous detection of various cellular components. A fast, sensitive and cheap method of the quantification of fixed adherent cells is described here. It is based on the incubation of DAPI- or Hoechst 33342-stained cells in a solution containing SDS. The presence of SDS results in the quick de-staining of DNA and simultaneously, in an up-to-1,000-fold increase of the fluorescence intensity of the used dyes. This increase can be attributed to the micelle formation of SDS. The method is sufficiently sensitive to reveal around 50-70 human diploid cells. It is compatible with immunocytochemical detections, the detection of DNA replication and cell cycle analysis by image cytometry. The procedure was successfully tested for the analysis of cytotoxicity. The method is suitable for the quantification of cells exhibiting low metabolic activity including senescent cells. The developed procedure provides high linearity and the signal is high for at least 20 days at room temperature. Only around 90 to 120 minutes is required for the procedure's completion.
- MeSH
- Staining and Labeling methods MeSH
- Cell Adhesion MeSH
- Cell Line MeSH
- Cell Cycle MeSH
- Cytophotometry methods MeSH
- Diploidy * MeSH
- DNA analysis chemistry MeSH
- Sodium Dodecyl Sulfate chemistry MeSH
- Fluorescent Dyes chemistry MeSH
- HeLa Cells MeSH
- Humans MeSH
- Cell Line, Tumor MeSH
- Cell Count instrumentation methods MeSH
- DNA Replication * MeSH
- Reproducibility of Results MeSH
- Cell Survival MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
This paper describes a comparative systems level analysis of the developmental proteome and transcriptome in the model antibiotic-producing eubacterium Streptomyces coelicolor, cultured on different media. The analysis formulates expression as the superposition of effects of regulatory networks and biological processes which can be identified using singular value decomposition (SVD) of a data matrix formed by time series measurements of expression of individual genes throughout the cell cycle of the bacterium. SVD produces linearly orthogonal factors, each of which can represent an independent system behavior defined by a linear combination of the genes/proteins highly correlated with the corresponding factor. By using SVD of the developmental time series of gene expression, as measured by both protein and RNA levels, we show that on the highest level of control (representing the basic kinetic behavior of the population), the results are identical, regardless of the type of experiment or cultivation method. The results show that this approach is capable of identifying basic regulatory processes independent of the environment in which the organism lives. It also shows that these processes are manifested equally on protein and RNA levels. Biological interpretation of the correlation of the genes and proteins with significant eigenprofiles (representing the highest level kinetic behavior of protein and/or RNA synthesis) revealed their association with metabolic processes, stress responses, starvation, and secondary metabolite production.
- MeSH
- Bacterial Proteins genetics metabolism MeSH
- RNA, Bacterial genetics metabolism MeSH
- Citric Acid Cycle MeSH
- Financing, Organized MeSH
- Data Interpretation, Statistical MeSH
- RNA, Messenger genetics metabolism MeSH
- Heat-Shock Proteins genetics metabolism MeSH
- Proteomics methods statistics & numerical data MeSH
- Gene Expression Profiling methods statistics & numerical data MeSH
- Streptomyces genetics metabolism growth & development MeSH
- Systems Biology MeSH
- Publication type
- Comparative Study MeSH
... Micro-Raman Characterisation of High and Low Spin Haem Moieties Within a Single 7 -- Living Red Blood Cell ... ... Toniolo a Vibrational Investigation of the Co(II)-Carnosine Oxygenated Complexes at Basic pH 35 -- A. ... ... Fastermann, 1 Heberle -- A New Method Coupling Polarized ATR-FTIR Spectroscopy and iH/^H Exchange to ... ... 60 -- Study the Dynamics of the Membrane Embedded Domain of LmrA During Its -- Catalytic Cycle -- V. ... ... Vicent -- Analysis of Ca and AI Polygalacturonate by Means of FT-Raman Spectroscopy 116 -- C. ...
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Maintenance of genome integrity is essential for homeostasis and survival as impaired DNA damage response (DDR) may predispose to grave pathologies such as neurodegenerative and immunodeficiency syndromes, cancer and premature aging. Therefore, accurate assessment of DNA damage caused by environmental or metabolic genotoxic insults is critical for contemporary biomedicine. The available physical, flow cytometry and sophisticated scanning approaches to DNA damage estimation each have some drawbacks such as insufficient sensitivity, limitation to analysis of cells in suspension, or high costs and demand for trained personnel. Here we present an option how to transform a regular fluorescence microscope and personal computer with common software into a functional alternative to high-throughput screening devices. In two detailed protocols we introduce a new semi-automatic procedure allowing for very sensitive, quantitative, rapid and simple fluorescence image analysis in thousands of adherent cells per day. Sensitive DNA breakage estimation through analysis of phosphorylated histone H2AX (gamma-H2AX), and homologous recombination (HR) assessed by a new RPA/Rad51 dual-marker approach illustrate the advantages and applicability of this technique. Our present data on assessment of low radiation doses, repair kinetics, spontaneous DNA damage in cancer cells, as well as constitutive and replication stress-induced HR events and their dependence on upstream factors within the DDR machinery document the versatility of the method. We believe this affordable approach may facilitate mechanistic insights into the role of low-dose DNA damage in human diseases, and generally promote both basic and translational research in many areas of biomedicine where suitable fluorescence markers are available.
Sugarcane (Saccharum spp.) is a globally important crop for sugar and bioenergy production. Its highly polyploid, complex genome has hindered progress in understanding its molecular structure. Flow cytometric sorting and analysis has been used in other important crops with large genomes to dissect the genome into component chromosomes. Here we present for the first time a method to prepare suspensions of intact sugarcane chromosomes for flow cytometric analysis and sorting. Flow karyotypes were generated for two S. officinarum and three hybrid cultivars. Five main peaks were identified and each genotype had a distinct flow karyotype profile. The flow karyotypes of S. officinarum were sharper and with more discrete peaks than the hybrids, this difference is probably due to the double genome structure of the hybrids. Simple Sequence Repeat (SSR) markers were used to determine that at least one allelic copy of each of the 10 basic chromosomes could be found in each peak for every genotype, except R570, suggesting that the peaks may represent ancestral Saccharum sub genomes. The ability to flow sort Saccharum chromosomes will allow us to isolate and analyse chromosomes of interest and further examine the structure and evolution of the sugarcane genome.
- MeSH
- Alleles MeSH
- Cell Cycle drug effects genetics MeSH
- Chromosomes, Plant genetics MeSH
- DNA, Plant metabolism MeSH
- Fluorescence MeSH
- Genome, Plant * MeSH
- Hydroxyurea pharmacology MeSH
- Karyotype MeSH
- Kinetics MeSH
- Plant Roots drug effects MeSH
- Polyploidy * MeSH
- Flow Cytometry methods MeSH
- Saccharum drug effects genetics MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Electromagnetic fields are usually absent in the picture of processes taking place in living cells which is dominated by biochemistry, molecular genetics and microscopic morphology. Yet experimental and theoretical studies suggest that this omission is not justified. At the end of 1960's H. Fröhlich elaborated a semi-phenomenological model of polar oscillating units that are metabolically driven, exchange energy with the cell's internal heat reservoir, and store part of the energy in excited vibrational modes in such way, that mode with the lowest frequency becomes highly excited, while the higher-order modes remain near thermal equilibrium. This affords energy-hungry chemical reactions to take place while the rest of the cell is not exposed to heat stress. At present, part of the cytoskeleton – microtubules – are deemed to fulfil the role of oscillating units. The paper provides an introduction to the Fröhlich ideas for readers with background in medicine and biology in that it avoids mathematical formulas and relies on figures to convey information about the basic properties of the model. The essential features of the Fröhlich model – most notably the energy condensation – are demonstrated on ensemble encompassing three coupled vibration modes that can be exactly described using original diagrammatic method.
- MeSH
- Adenosine Triphosphate metabolism MeSH
- Biomedical Research methods trends MeSH
- Electromagnetic Phenomena MeSH
- Energy Metabolism physiology MeSH
- Financing, Organized MeSH
- Photosynthesis physiology MeSH
- Cell Physiological Phenomena physiology genetics MeSH
- Guanosine Triphosphate metabolism MeSH
- Humans MeSH
- Microtubules physiology metabolism MeSH
- Mitochondria physiology metabolism MeSH
- Cell Wall Skeleton physiology chemistry metabolism MeSH
- Statistics as Topic MeSH
- Models, Theoretical MeSH
- Check Tag
- Humans MeSH
... Shields: Renaissance in lymphology XIV -- Basic Science -- K. B. Roberts y J. A. ... ... Casley-Smith: Colloidal osmotic pressure and the initial lymphatic cycle 33 -- ?. R. ... ... Hauck: Cathodo-luminescence method in the scanning electron microscope applied to basic lymphology 47 ... ... Olszewski: Cell subpopulations in prenodal lymph of normal dogs 141 -- G. S. Del Giacco, A. ... ... receptors for IgG and of phagocytic cells 157 -- H. ...
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