搜索结果：DNA and cell biology

The 40,000-fold variation in eukaryote haploid DNA content is unrelated to organismic complexity or to the numbers of protein-coding genes. In eukaryote microorganisms, as well as in animals and plants, DNA content is strongly correlated with cell volume and nuclear volume, and with cell cycle length and minimum generation time. These correlations are simply explained by postulating that DNA has 2 major functions unrelated to its protein-coding capacity: (1) the control of cell volume by the number of replicon origins, and (2) the determination of nuclear volume by the overall bulk of the DNA: cell growth rates are determined by the cell volume and by the area of the nuclear envelope available for nucleocytoplasmic transport of RNA, which in turn depends on the nuclear volume and therefore on the DNA content. During evolution nuclear volume, and therefore DNA content, has to be adjusted to the cell volume to allow reasonable growth rates. The great diversity of cell volumes and growth rates, and therefore of DNA contents, among eukaryotes results from a varying balance in different species between r-selection, which favours small cells and rapid growth rates and therefore low DNA C-values, and K-selection which favours large cells and slow growth rates and therefore high DNA C-values. In multicellular organisms cell size needs to vary in different tissues: size differences between somatic cells result from polyteny, endopolyploidy, or the synthesis of nucleoskeletal RNA. Conflict between the need for large ova and small somatic cells explains why lampbrush chromosomes, nurse cells, chromatin diminution and chromosome elimination evolved. Similar evolutionary considerations clarify the nature of polygenes, the significance of the distribution of haploidy, diploidy and dikaryosis in life cycles and of double fertilization in angiosperms, and of heteroploidy despite DNA constancy in cultured cells, and other puzzles in eukaryote chromosome biology. Eukaryote DNA can be divided into genic DNA (G-DNA), which codes for proteins (or serves as recognition sites for proteins involved in transcription, replication and recombination), and nucleoskeletal DNA (S-DNA) which exists only because of its nucleoskeletal role in determining the nuclear volume (which it shares with G-DNA, and performs not only directly, but also indirectly by coding for nucleoskeletal RNA). Mechanistic and evolutionary implications of this are discussed.

Contents of Volume 1: Part A. Cell and Tissue Culture: Associated Techniques. Section 1. General Techniques. Section 2. Culture of Specific Cell Types: Stem Cells. Section 3. Culture of Specific Cell Types: Haemopoietic, Mesenchymal, and Epithelial. Section 4. Differentiation and Reprogramming of Somatic Cells. Section 5. Immortalisation. Section 6. Somatic Cell Hybrids. Section 7. Cell Separation Techniques. Section 8. Cell Cycle Analysis. Section 9. Cytotoxic and Cell Growth Assays. Section 10. Apoptosis. Section 11. Assays of Cell Transformation, Tumorigenesis, Invasion and Wound Healing. Section 12. Electrophysiological Methods. Section 13. Organ Cultures. Part B. Viruses. Section 14. Growth and Purification of Viruses. Part C. Antibodies. Section 15. Production and Purification of Antibodies. Part D. Immunocytochemistry. Section 16. Immunofluoresence. Part E. Appendix. Contents of Volume 2: Part A. Organelles and Cellular Structures. Section 1. Isolation: Plasma Membrane, Organelles, and Cellular Structures. Section 2. Vital Staining of Cells/Organelles. Section 3. Protein Purification. Part B. Assays. Section 4. Endocytic and Exocytic Pathways. Section 5. Membranes. Section 6. Mitochondria. Section 7. Nuclear Transport. Section 8. Chromatin Assembly. Section 9. Signal Transduction Assays. Section 10. Assays and Models of in Vitro and in Vitro Motility. Section 11. Mechanical Stress in Single Cells. Part C. Appendix. Contents of Volume 3. Part A. Imaging Techniques. Section 1. Light Microscopy. Section 2. Digital Video Microscopy. Section 3. Confocal Microscopy of Living Cells and Fixed Cells. Section 4. Fluorescent Microscopy of Living Cells. Section 5. Use of Fluorescent Dyes for Studies of Intracellular Physiological Parameters. Section 6. Digital Image Processing, Analysis, Storage, and Display. Part B. Electron Microscopy. Section 7. Specimen Preparation Techniques. Section 8. Cryotechniques. Section 9. Electron Microscopy Studies of the Cytoskeleton. Section 10. Immunoelectron Microscopy. Part C. Scanning Probe and Scanning Electron Microscopy. Section 11. Scanning Probe and Scanning Electron Microscopy. Part D. Microdissection. Section 12. Tissue and Chromosome Microdissection. Part E. Tissue Arrays. Section 13. Tissue Arrays. Part F. Cytogenetics and in Situ Hybridisation Section 14. Cytogenetics. Section 15. In Situ Hybridisation. Part G. Genomics. Section 16. Genomics. Part H. Transgenic, Knockouts, and Knockdown Methods. Section 17. Transgenic, Knockouts and knock-down methods. Contents of Volume 4. Part A. Transfer of Macromolecules. Section 1. Proteins. Section 2. Genes. Section 3. Somatic Cell Nuclear Transfer. Part B. Expression Systems. Section 4. Expression Systems. Part C. Gene Expression Profiling. Section 5. Differential Gene Expression. Part D. Proteins. Section 6. Protein Determination and Analysis. Section 7. Sample Fractionation for Proteomics. Section 8. gel Electrophoresis. Section 9. Detection of Proteins in Gels. Section 10. Gel Profiling of Posttranslationally Modified Proteins. Section 11. Protein/Protein and Protein/Small Molecule Interactions. Section 12. Functional Proteomics. Section 13. Protein/DNA Interactions. Section 14. Protein Degradation. Section 15. Mass Spectrometry: Protein Identification and Interactions. Part E. Appendix.

Eukaryotic cells make many types of primary and processed RNAs that are found either in specific subcellular compartments or throughout the cells. A complete catalogue of these RNAs is not yet available and their characteristic subcellular localizations are also poorly understood. Because RNA represents the direct output of the genetic information encoded by genomes and a significant proportion of a cell’s regulatory capabilities are focused on its synthesis, processing, transport, modification and translation, the generation of such a catalogue is crucial for understanding genome function. Here we report evidence that three-quarters of the human genome is capable of being transcribed, as well as observations about the range and levels of expression, localization, processing fates, regulatory regions and modifications of almost all currently annotated and thousands of previously unannotated RNAs. These observations, taken together, prompt a redefinition of the concept of a gene. A description is given of the ENCODE effort to provide a complete catalogue of primary and processed RNAs found either in specific subcellular compartments or throughout the cell, revealing that three-quarters of the human genome can be transcribed, and providing a wealth of information on the range and levels of expression, localization, processing fates and modifications of known and previously unannotated RNAs. These authors describe the ENCODE (Encyclopedia of DNA Elements) effort to provide a complete catalogue of primary and processed RNAs found either in specific sub-cellular compartments or throughout the cell. They show that three-quarters of the human genome can be transcribed, and provide a wealth of information about the range and levels of expression, localization, processing fates and modifications of both known and previously unannotated RNAs. Collectively, these observations suggest that the current concept of a gene should be revisited.

The terms MSC and MSCs have become the preferred acronym to describe a cell and a cell population of multipotential stem/progenitor cells commonly referred to as mesenchymal stem cells, multipotential stromal cells, mesenchymal stromal cells, and mesenchymal progenitor cells. The MSCs can differentiate to important lineages under defined conditions in vitro and in limited situations after implantation in vivo. MSCs were isolated and described about 30 years ago and now there are over 55,000 publications on MSCs readily available. Here, we have focused on human MSCs whenever possible. The MSCs have broad anti-inflammatory and immune-modulatory properties. At present, these provide the greatest focus of human MSCs in clinical testing; however, the properties of cultured MSCs in vitro suggest they can have broader applications. The medical utility of MSCs continues to be investigated in over 950 clinical trials. There has been much progress in understanding MSCs over the years, and there is a strong foundation for future scientific research and clinical applications, but also some important questions remain to be answered. Developing further methods to understand and unlock MSC potential through intracellular and intercellular signaling, biomedical engineering, delivery methods and patient selection should all provide substantial advancements in the coming years and greater clinical opportunities. The expansive and growing field of MSC research is teaching us basic human cell biology as well as how to use this type of cell for cellular therapy in a variety of clinical settings, and while much promise is evident, careful new work is still needed.

BACKGROUND: It is not yet known whether DNA methylation levels can be used to accurately predict age across a broad spectrum of human tissues and cell types, nor whether the resulting age prediction is a biologically meaningful measure. RESULTS: I developed a multi-tissue predictor of age that allows one to estimate the DNA methylation age of most tissues and cell types. The predictor, which is freely available, was developed using 8,000 samples from 82 Illumina DNA methylation array datasets, encompassing 51 healthy tissues and cell types. I found that DNA methylation age has the following properties: first, it is close to zero for embryonic and induced pluripotent stem cells; second, it correlates with cell passage number; third, it gives rise to a highly heritable measure of age acceleration; and, fourth, it is applicable to chimpanzee tissues. Analysis of 6,000 cancer samples from 32 datasets showed that all of the considered 20 cancer types exhibit significant age acceleration, with an average of 36 years. Low age-acceleration of cancer tissue is associated with a high number of somatic mutations and TP53 mutations, while mutations in steroid receptors greatly accelerate DNA methylation age in breast cancer. Finally, I characterize the 353 CpG sites that together form an aging clock in terms of chromatin states and tissue variance. CONCLUSIONS: I propose that DNA methylation age measures the cumulative effect of an epigenetic maintenance system. This novel epigenetic clock can be used to address a host of questions in developmental biology, cancer and aging research.

When mammalian cell cultures or mice are exposed to ionizing radiation in survivable or lethal amounts, novel mass components are found in the histone H2A region of two-dimensional gels. Collectively referred to as gamma, these components are formed in vivo by several procedures that introduce double-stranded breaks into DNA. gamma-Components, which appeared to be the only major novel components detected by mass or 32PO4 incorporation on acetic acid-urea-Triton X-100-acetic acid-urea-cetyltrimethylammonium bromide or SDS-acetic acid-urea-cetyltrimethylammonium bromide gels after exposure of cells to ionizing radiation, are shown to be histone H2AX species that have been phosphorylated specifically at serine 139. gamma-H2AX appears rapidly after exposure of cell cultures to ionizing radiation; half-maximal amounts are reached by 1 min and maximal amounts by 10 min. At the maximum, approximately 1% of the H2AX becomes gamma-phosphorylated per gray of ionizing radiation, a finding that indicates that 35 DNA double-stranded breaks, the number introduced by each gray into the 6 x 10(9) base pairs of a mammalian G1 genome, leads to the gamma-phosphorylation of H2AX distributed over 1% of the chromatin. Thus, about 0.03% of the chromatin appears to be involved per DNA double-stranded break. This value, which corresponds to about 2 x 10(6) base pairs of DNA per double-stranded break, indicates that large amounts of chromatin are involved with each DNA double-stranded break. Thus, gamma-H2AX formation is a rapid and sensitive cellular response to the presence of DNA double-stranded breaks, a response that may provide insight into higher order chromatin structures.

Emerging evidence is revealing that exosomes contribute to many aspects of physiology and disease through intercellular communication. However, the biological roles of exosome secretion in exosome-secreting cells have remained largely unexplored. Here we show that exosome secretion plays a crucial role in maintaining cellular homeostasis in exosome-secreting cells. The inhibition of exosome secretion results in the accumulation of nuclear DNA in the cytoplasm, thereby causing the activation of cytoplasmic DNA sensing machinery. This event provokes the innate immune response, leading to reactive oxygen species (ROS)-dependent DNA damage response and thus induce senescence-like cell-cycle arrest or apoptosis in normal human cells. These results, in conjunction with observations that exosomes contain various lengths of chromosomal DNA fragments, indicate that exosome secretion maintains cellular homeostasis by removing harmful cytoplasmic DNA from cells. Together, these findings enhance our understanding of exosome biology, and provide valuable new insights into the control of cellular homeostasis.

BACKGROUND: There has been a long-standing need in biomedical research for a method that quantifies the normally mixed composition of leukocytes beyond what is possible by simple histological or flow cytometric assessments. The latter is restricted by the labile nature of protein epitopes, requirements for cell processing, and timely cell analysis. In a diverse array of diseases and following numerous immune-toxic exposures, leukocyte composition will critically inform the underlying immuno-biology to most chronic medical conditions. Emerging research demonstrates that DNA methylation is responsible for cellular differentiation, and when measured in whole peripheral blood, serves to distinguish cancer cases from controls. RESULTS: Here we present a method, similar to regression calibration, for inferring changes in the distribution of white blood cells between different subpopulations (e.g. cases and controls) using DNA methylation signatures, in combination with a previously obtained external validation set consisting of signatures from purified leukocyte samples. We validate the fundamental idea in a cell mixture reconstruction experiment, then demonstrate our method on DNA methylation data sets from several studies, including data from a Head and Neck Squamous Cell Carcinoma (HNSCC) study and an ovarian cancer study. Our method produces results consistent with prior biological findings, thereby validating the approach. CONCLUSIONS: Our method, in combination with an appropriate external validation set, promises new opportunities for large-scale immunological studies of both disease states and noxious exposures.

A cell's behavior is a consequence of the complex interactions between its numerous constituents, such as DNA, RNA, proteins and small molecules. Cells use signaling pathways and regulatory mechanisms to coordinate multiple processes, allowing them to respond to and adapt to an ever-changing environment. The large number of components, the degree of interconnectivity and the complex control of cellular networks are becoming evident in the integrated genomic and proteomic analyses that are emerging. It is increasingly recognized that the understanding of properties that arise from whole-cell function require integrated, theoretical descriptions of the relationships between different cellular components. Recent theoretical advances allow us to describe cellular network structure with graph concepts and have revealed organizational features shared with numerous non-biological networks. We now have the opportunity to describe quantitatively a network of hundreds or thousands of interacting components. Moreover, the observed topologies of cellular networks give us clues about their evolution and how their organization influences their function and dynamic responses.

Genetic changes underlying clear cell renal cell carcinoma (ccRCC) include alterations in genes controlling cellular oxygen sensing (for example, VHL) and the maintenance of chromatin states (for example, PBRM1). We surveyed more than 400 tumours using different genomic platforms and identified 19 significantly mutated genes. The PI(3)K/AKT pathway was recurrently mutated, suggesting this pathway as a potential therapeutic target. Widespread DNA hypomethylation was associated with mutation of the H3K36 methyltransferase SETD2, and integrative analysis suggested that mutations involving the SWI/SNF chromatin remodelling complex (PBRM1, ARID1A, SMARCA4) could have far-reaching effects on other pathways. Aggressive cancers demonstrated evidence of a metabolic shift, involving downregulation of genes involved in the TCA cycle, decreased AMPK and PTEN protein levels, upregulation of the pentose phosphate pathway and the glutamine transporter genes, increased acetyl-CoA carboxylase protein, and altered promoter methylation of miR-21 (also known as MIR21) and GRB10. Remodelling cellular metabolism thus constitutes a recurrent pattern in ccRCC that correlates with tumour stage and severity and offers new views on the opportunities for disease treatment. The Cancer Genome Atlas Research Network reports an integrative analysis of more than 400 samples of clear cell renal cell carcinoma based on genomic, DNA methylation, RNA and proteomic characterisation; frequent mutations were identified in the PI(3)K/AKT pathway, suggesting this pathway might be a potential therapeutic target, among the findings is also a demonstration of metabolic remodelling which correlates with tumour stage and severity. The Cancer Genome Atlas consortium reports an integrative analysis of more than 400 samples of clear cell renal carcinoma on the basis of genomic, DNA methylation, RNA and proteomic characterization. The data reveal frequent mutations in the PI(3)K/AKT pathway, suggesting that this pathway might be a potential therapeutic target, in addition to an array of epigenetic alterations that are linked to specific mutations in chromatin-associated proteins. One notable finding is the presence of a metabolic shift in aggressive cancers, correlating with tumour stage and severity.

Infiltrating stromal and immune cells form the major fraction of normal cells in tumour tissue and not only perturb the tumour signal in molecular studies but also have an important role in cancer biology. Here we describe ‘Estimation of STromal and Immune cells in MAlignant Tumours using Expression data’ (ESTIMATE)—a method that uses gene expression signatures to infer the fraction of stromal and immune cells in tumour samples. ESTIMATE scores correlate with DNA copy number-based tumour purity across samples from 11 different tumour types, profiled on Agilent, Affymetrix platforms or based on RNA sequencing and available through The Cancer Genome Atlas. The prediction accuracy is further corroborated using 3,809 transcriptional profiles available elsewhere in the public domain. The ESTIMATE method allows consideration of tumour-associated normal cells in genomic and transcriptomic studies. An R-library is available on https://sourceforge.net/projects/estimateproject/ . Tumour biopsies contain contaminating normal cells and these can influence the analysis of tumour samples. In this study, Yoshihara et al.develop an algorithm based on gene expression profiles from The Cancer Genome Atlas to estimate the number of contaminating normal cells in tumour samples.

PART : INTRODUCTION. - An Overview of Cells and Cell Research - The Chemistry of Cells - Fundamentals of Molecular Biology - PART I1: THE FLOW OF GENETIC INFORMATION - The Organization and Sequences of Cellular Genomes - Replication, Maintenance, and Rearrangements of Genomic DNA - RNA Synthesis and Processing - Protein Synthesis, Processing, and Regulation - PART III: CELL STRUCTURE AND FUNCTION - The Nucleus - Protein Sorting and Transport: The Endoplasmic Reticulum, Golgi - Apparatus, and Lysosomes - Bioenergetics and Metabolism: Mitochondria, Chloroplasts, and - Peroxisomes - The Cytoskeleton and Cell Movement - The Cell Surface - PART IV CELL REGULATION - Cell Signaling - The Cell Cycle - Cancer -

Part I: Molecular Biology of Cancer Molecular Methods in Oncology Section 1. Amplification Techniques Section 2. RNA Interference Section 3. cDNA arrays Section 4. Tissue arrays Section 5. Cytogenetics Section 6. Bioinformatics Genomics and Proteomics Molecular Targets in Oncology Section 1. Signal transduction systems Section 2. Cell cycle Section 3. Apoptosis Section 4. Telomerase Invasion and Metastases Angiogenesis Cancer Immunology Part II: Principles of Oncology Etiology of Cancer: Viruses Section 1. RNA Viruses Section 2. DNA Viruses Etiology of Cancer: Chemical Factors Etiology of Cancer: Tobacco Etiology of Cancer: Physical Factors Epidemiology of Cancer Section 1. Epidemiologic Methods Section 2. Cancer Statistics Principles of Surgical Oncology Section 1. General Issues Section 2. Laparascopic Surgery Principles of Radiation Oncology Principles of Medical Oncology Pharmacology of Cancer Chemotherapy Section 2. Pharmocokinetics Section 3. Pharmacogenomics Section 4. Alkylating Agents Section 5. Cisplatin and its Analogues Section 6. Antimetabolites Section 7. Topoisomerase Interactive Agents Section 8. Antimicrotubule Agents Section 9. Miscellaneous Chemotherapeutic Agents Pharmacology of Cancer Biotherapeutics Section 1. Interferon Section 2. Interleukin 2 Section 3. Histone deacetylase inhibitors as differentiation agents Section 4. Monoclonal Antibodies Pharmacology of Endocrine Manipulation Design and Analysis of Clinical Trials Part III: Practice of Oncology Cancer Prevention: Preventing Tobacco-Related Cancers Cancer Prevention: Diet and Chemopreventive Agents Section 1. Dietary fat Section 2. Dietary Fiber Section 3. Dietary fruits and vegetables: naturally occurring anticarcinogens Section 4. Retinoids, carotenoids and micronutrients Section 5. Dietary Carcinogens Section 6. Cyclo-oxygenase inhibitors Section 7. Physical Activity and Body Weight Cancer Prevention: Role of Surgery in Cancer Prevention Cancer Screening Advanced Molecular Diagnostics Advanced Imaging Methods Section 1. Functional and Metabolic Imaging Section 2. Interventional Radiology Cancer Diagnosis: Endoscopy Section 1. Gastrointestinal endoscopy Section 2. Respiratory Tract Cancer of the Head and Neck Section 1. Molecular Biology of Head and Neck Tumors Section 2. Treatment of Head and Neck Cancers Section 3. Rehabilitation after Treatment for Head Cancer of the Lung Section 1. Molecular Biology of Lung Cancer Section 2. Non-small Cell Lung Cancer Section 3. Small Cell Lung Cancer Neoplasms of the Mediastinum Cancers of the Gastrointestinal Tract

Humans circulate quadrillions of exosomes at all times. Exosomes are a class of extracellular vesicles released by all cells, with a size range of 40-150 nm and a lipid bilayer membrane. Exosomes contain DNA, RNA, and proteins. Exosomes likely remove excess and/or unnecessary constituents from the cells, functioning like garbage bags, although their precise physiological role remains unknown. Additionally, exosomes may mediate specific cell-to-cell communication and activate signaling pathways in cells they fuse or interact with. Exosomes are detected in the tumor microenvironment, and emerging evidence suggests that they play a role in facilitating tumorigenesis by regulating angiogenesis, immunity, and metastasis. Circulating exosomes can be used as liquid biopsies and noninvasive biomarkers for early detection, diagnosis, and treatment of cancer patients.

Biologists can now prepare and image thousands of samples per day using automation, enabling chemical screens and functional genomics (for example, using RNA interference). Here we describe the first free, open-source system designed for flexible, high-throughput cell image analysis, CellProfiler. CellProfiler can address a variety of biological questions quantitatively, including standard assays (for example, cell count, size, per-cell protein levels) and complex morphological assays (for example, cell/organelle shape or subcellular patterns of DNA or protein staining).

Programmed cell death (PCD) plays a key role in developmental biology and in maintenance of the steady state in continuously renewing tissues. Currently, its existence is inferred mainly from gel electrophoresis of a pooled DNA extract as PCD was shown to be associated with DNA fragmentation. Based on this observation, we describe here the development of a method for the in situ visualization of PCD at the single-cell level, while preserving tissue architecture. Conventional histological sections, pretreated with protease, were nick end labeled with biotinylated poly dU, introduced by terminal deoxy-transferase, and then stained using avidin-conjugated peroxidase. The reaction is specific, only nuclei located at positions where PCD is expected are stained. The initial screening includes: small and large intestine, epidermis, lymphoid tissues, ovary, and other organs. A detailed analysis revealed that the process is initiated at the nuclear periphery, it is relatively short (1-3 h from initiation to cell elimination) and that PCD appears in tissues in clusters. The extent of tissue-PCD revealed by this method is considerably greater than apoptosis detected by nuclear morphology, and thus opens the way for a variety of studies.

MOTIVATION: The recently released Infinium HumanMethylation450 array (the '450k' array) provides a high-throughput assay to quantify DNA methylation (DNAm) at ∼450 000 loci across a range of genomic features. Although less comprehensive than high-throughput sequencing-based techniques, this product is more cost-effective and promises to be the most widely used DNAm high-throughput measurement technology over the next several years. RESULTS: Here we describe a suite of computational tools that incorporate state-of-the-art statistical techniques for the analysis of DNAm data. The software is structured to easily adapt to future versions of the technology. We include methods for preprocessing, quality assessment and detection of differentially methylated regions from the kilobase to the megabase scale. We show how our software provides a powerful and flexible development platform for future methods. We also illustrate how our methods empower the technology to make discoveries previously thought to be possible only with sequencing-based methods. AVAILABILITY AND IMPLEMENTATION: http://bioconductor.org/packages/release/bioc/html/minfi.html. CONTACT: khansen@jhsph.edu; rafa@jimmy.harvard.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The packaging of chromosomal DNA by nucleosomes condenses and organizes the genome, but occludes many regulatory DNA elements. However, this constraint also allows nucleosomes and other chromatin components to actively participate in the regulation of transcription, chromosome segregation, DNA replication, and DNA repair. To enable dynamic access to packaged DNA and to tailor nucleosome composition in chromosomal regions, cells have evolved a set of specialized chromatin remodeling complexes (remodelers). Remodelers use the energy of ATP hydrolysis to move, destabilize, eject, or restructure nucleosomes. Here, we address many aspects of remodeler biology: their targeting, mechanism, regulation, shared and unique properties, and specialization for particular biological processes. We also address roles for remodelers in development, cancer, and human syndromes.