细胞功能测定

ADP-ribosylation factor (Arf) proteins were first identified as cofactors for cholera toxin-catalyzed ADP-ribosylation of the heterotrimeric G-protein Gs. Subsequent cloning led to the discovery that Arfs were part of a group of GTP-binding proteins that is a subfamily of the ras supe ...

TheADP-ribosylation factor proteins (ARFs) are ~20-kDa guanine-nucleotidebinding proteins found in all eukaryotic cells, which are a critical part of the minimum machinery required for vesicle formation at the Golgi and other membranes (1–3). The six mammalian ARFs have been grouped ...

The Arf family of GTP-binding proteins is highly conserved and widely expressed in eukaryotic cells. Arfs can be divided into three classes based on amino-acid sequence. Class I Arfs include mammalian Arfs 1-3, Class II includes Arfs 4, and 5, and Class III Arfs have only one member-Arf6 (1). All Arf prote ...

ARF-family GTPases function as regulators of multiple membrane trafficking processes in all eukaryotic cells. Six ARF proteins have been identified in humans, all of which contain a myristoyl residue, attached to their aminoterminal glycine by an amide bond. In their active, guanosine t ...

Nucleocytoplasmic transport occurs through nuclear pore complexes (NPC), macromolecular structures which span the nuclear envelope (1). NPCs contain aqueous channels with a diameter of ~9 nm, allowing ions, metabolites, and small proteins to passively diffuse between the nucle ...

Nucleocytoplasmic transport of protein is central to the regulation of many cellular functions. Nuclear import and export takes place through nuclear pore complexes (NPCs; reviewed in refs. 1 and 2), large supramolecular structures that span the nuclear envelope. Cargo proteins cont ...

Ran is a GTPase of the Ras superfamily. Until recently, its primary role was believed to be in interphase, regulating nucleocytoplasmic transport (1). However, recent studies have identified additional roles for Ran in regulating spindle assembly and maintenance in mitosis (2-10). In add ...

During the last two decades since the first oncogene product called v-Src was identified as a constitutively activated mutant of a normal mitogenic gene (proto-oncogene) encoding a Tyr kinase called c-Src in early 1980s, it was firmly established that the malignant transformation of norm ...

The investigation into the relationship between reactive oxygen species (ROS) and the signaling pathways of the Ras family of small GTPases has experienced a recent surge in interest. Known to cause DNA damage, and implicated in disease processes as wide-ranging as atherosclerosis and ki ...

A variety of techniques can be used to find protein partners, including immunoprecipitation, affinity chromatography, blot overlays, and yeast twohybrid screening. Of these, the blot overlay protocol of sodium dodecyl sulfate (SDS)-acrylamide-gel-separated proteins provi ...

Rho-like GTPases, including RhoA, Rac1 and Cdc42, have been implicated in the control of a wide range of biological processes such as the organization of cytoskeletal structures, adhesion, motility, transcriptional activation, and cell-cycle progression (1–3). The Rho-like GTPases ...

Gene-specific disruption and protein-specific inhibition techniques are powerful tools for the study of protein functions in vivo. A number of methods have been applied to investigate the loss of protein function. These include generation of knockout animals or cell lines, applicat ...

In living bodies, there are many cell-cell interactions, including epithelial-mesenchymal interactions, to control cell growth and differentiation, and disturbances to such systems are believed to be a critical reason for many diseases, including cancer. Recently, many growth f ...

This chapter deals with experimental protocols and considerations related to the culture of epithelial cells under anchorage-independent conditions in liquid media. This technique has proven to be a powerful tool in studying the effects of loss of extracellular matrix interaction ...

In this chapter, we describe an organ culture method that allows skin tissues isolated from 12.5- or 13.5-d postcoitus mouse embryos to develop in a manner histologically and temporally similar to the process in vivo. In this culture system, epidermal growth factor specifically and complete ...

In this chapter, we present technical details for the generation of in vitro skin equivalents consisting of collagen gels with incorporated fibroblasts covered by proliferating and differentiating keratinocytes. Epithelial-mesenchymal interactions are clearly manif ...

In vitro fabrication of human epidermal tissues that mimic the biochemical and morphologic properties of human skin, known as skin-equivalent (organotypic) cultures, has opened new avenues in the study of skin biology. In this chapter, methods for the generation of these tissues from their ...

Recent work from our laboratory has led to the development and validation of fluorescence-activated cell sorting (FACS)-based techniques to prospectively isolate viable keratinocyte stem cells from both human and murine skin. Here we describe a step-by-step method to apply our techn ...

It is well accepted that homeostasis of continuously renewing adult tissues, such as the epidermis, is maintained by somatic stem cells. These are undifferentiated, self-renewing cells, which also produce daughter transit amplifying (TA) cells that make up the majority of the prolifer ...

The skin is a dynamic tissue in which terminally differentiated keratinocytes are replaced by the proliferation of new epithelial cells that will undergo differentiation. The rapid and continual turnover of skin throughout life depends on a cell population with unique characteris ...