细胞技术

The polymerase chain reaction (PCR) has revolutionized molecular biology. Portions of single-copy per cell genes (and cDNAs) prepared from very small tissue or cell samples can be specifically amplified for use in sequence determination, gene identification, and quantitation. Im ...

This brief guide is not intended as a full explanation of the theory and practice of nuclear magnetic resonance (NMR), on which there are a large number of excellent texts (1–3), but as an introduction to the terms used in the subsequent chapters. The section as a whole does not provide a comprehensive outli ...

Nuclear magnetic resonance (NMR) spectroscopy has become established in recent years as a uniquely powerful technique for studying the structures of proteins in solution. In a 1H spectrum, each hydrogen atom in the molecule gives rise to an individual signal, and in favorable cases, it is poss ...

Developing nerve growth factor (NGF)-dependent sympathetic neurons are one of the best-studied in vitro models of neuronal apoptosis and have been used to identify key components of the neuronal cell death pathway. This chapter describes how to prepare purified cultures of primary symp ...

Mitogen-activated protein kinases (MAPKs) are activated by a wide variety of cellular stimuli and involved in the regulation of most, if not all, cellular processes. Among them, the c-Jun N-terminal kinase and p38 MAPKs are predominantly induced in response to proinflammatory cytokines a ...

Developing sympathetic neurons, which depend on nerve growth factor for survival, are one of the best studied in vitro models of neuronal apoptosis and have been extensively used for cellular and molecular studies of the neuronal death pathway. Important apoptotic events after nerve gro ...

Methods for the detection of programmed cell death (PCD) in plants are reviewed with references for different biochemical, microscopic, and molecular assays. A detailed description of three different methods for the detection of biotic or abiotic PCD in plant tissues is included. The rea ...

Drosophila has unique genetic and cell biological advantages as a model system for the study of apoptosis. Many cell death genes are evolutionarily conserved between flies and mammals. Cell death can be induced by environmental stimuli and normally occurs during diverse developmental ...

The yeast two-hybrid system is a powerful technique for identifying proteins that interact with a specific protein of interest. The rationale of the yeast two-hybrid system relies on the physical separation of the DNA-binding domain from the transcriptional activation domain of sever ...

The phagocytic clearance of apoptotic cells is a highly efficient and non-phlogistic process in vivo. Research in this area has been limited, at least in part, by technical difficulties associated with the techniques used in the detailed study of apoptotic cell clearance mechanisms. This c ...

Caspases are cysteine proteases that play an essential role during apoptotic cell death and inflammation. They are synthesized as catalytically dormant proenzymes, containing an N-terminal prodomain, a large subunit (p20) containing the active site cysteine, and a small subunit (p ...

Apoptosis is frequently triggered by events that alter the expression of key target genes. Under these circumstances, the genes involved can be identified by techniques that analyze gene expression. Researchers now have a choice of reliable and effective methods for differential gene ...

Classical apoptotic cell death can be defined by certain morphological and biochemical characteristics that distinguish it from other forms of cell death. One such feature, which is a hallmark of apoptosis, is DNA fragmentation. In dying cells, DNA is cleaved by an endonuclease that fragme ...

This chapter describes a retroviral insertion mutagenesis approach using replication-deficient myeloproliferative sarcoma virus retroviral vectors to identify apoptosis regulatory genes in the interleukin-3-dependent Baf-3 cell line. We describe the retroviral i ...

Apoptosis is an active form of cell death that plays a critical role in lymphocyte development, selection and homeostasis. This process is characterized by the activation of biochemical pathways that lead to changes in cellular morphology (including cell shrinkage, membrane blebbi ...

Cysteine proteases of the caspase family play key roles in the execution of apoptosis and in the maturation of proinflammatory cytokines. During apoptosis signaling, the latent forms of caspase precursors undergo rapid proteolytic processing and activation. Thus, the measurement ...

Various techniques exist for the identification of apoptosis in tissue sections or intact cells. The use of simple morphology, electron microscopy, DNA-end labeling techniques, and immunochemical methods are reviewed, with a particular emphasis on in situ end-labeling. The analys ...

Mitochondrial membrane permeabilization (MMP) constitutes an early event of the apoptotic process. MMP affects both mitochondrial membranes. Inner MMP leads to the dissipation of the inner transmembrane potential and outer MMP culminates in the efflux of apoptogenic factors. The ...

Many cell cycle regulatory proteins have been shown to be able to regulate cell death. Activation of Cdk2 has been shown to be necessary for the apoptosis of quiescent cells such as thymocytes, neurons, and endothelial cells. This activation is stimulus-specific because it occurs in glucocor ...

The role of Ca2+ changes in the commitment to apoptosis has been appreciated for more than two decades. However, early work focused on increases in cytosolic Ca2+ levels that may not be associated with most examples of programmed cell death. Rather, recent studies indicate that release of Ca2+ from t ...