细胞功能测定

Since the discovery of the membrane-type matrix metalloproteinases (MT-MMPs) which are characterized by the existence of a C-terminal membrane-spanning domain followed by a short cytoplasmic “tail” (1) there has been much interest in the production of recombinant protein to facili ...

Deciding what system to use for recombinant matrix metalloproteinase (MMP) expression is often a matter of practicality (i.e., simplicity, cost effectiveness, time considerations). Of course, the final judgment of practicality is determined by the production of sufficient quant ...

E. coli is a convenient host in which to express recombinant proteins. The technology is available to most laboratories as it is relatively inexpensive and does not require extensive expertise. The major drawback of E. coli as an expression host is the inability of the organism to carry out many postt ...

Structural analysis of any protein, whether by X-ray crystallography or NMR, requires a reliable source of large amounts of good quality protein. Most proteins are not sufficiently abundant in their natural state to be used as the primary source, and so it is essential for recombinant protein to be ...

Currently more than twenty different matrix metalloproteinases (MMPs) and four tissue inhibitors of metalloproteinases (TIMPs) have been identified (1,2) by cDNA cloning. This chapter describes methods to purify collagenases (MMP-1, MMP-8, and MMP-13), gelatinases (MMP-2 and MMP ...

With the advent of recombinant DNA technology, numerous systems have been utilized for the overexpression of proteins. Recombinant protein expression in Escherichia coli (E. coli) typically provides large quantities of the protein of interest in a relatively short period of time. The e ...

Resident cells of tissues are capable of secreting an array of structurally related zinc endopeptidases known as matrix metalloproteinases (MMPs). They initiate the degradation of the surrounding macromolecules of the extracellular matrix (ECM), mostly proteoglycans and spe ...

Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) is a sensitive and rapid method to monitor the expression of specific mRNAs. Here we describe two approaches to quantification of steady-state levels of Matrix Metalloproteinase (MMP) and Tissue Inhibitor of Metallopro ...

Many metalloproteinase genes are transcriptionally regulated by differentiation, growth factors, cytokines, and hormones. Nuclear run-off assays and analysis of heterogeneous nuclear RNA (hnRNA) levels are two methods for studying changes in gene transcription. In this cha ...

The activity of matrix metalloproteinases (MMPs) is closely regulated by tissue inhibitors of metalloproteinases (TIMPs) under pathophysiological conditions. The quantitative imbalance between MMPs and TIMPs in tissues and body fluids is considered to cause tumor invasion, ...

The imminent completion of the human and mouse genome projects will reveal all metalloproteases and metalloprotease inhibitors such as those belonging to the MMP, TIMP, ADAM, and ADAMTS (1) families. At the present time, there are over 65 published genes in these families and it is very likely that t ...

Immunohistochemical techniques are a convenient method to identify the cells responsible for the production of MMPs and TIMPs in local tissues under pathophysiological conditions. Direct and indirect methods are presented for immunohistochemistry, but the latter is usually u ...

The degradation of the extracellular matrix during development and in disease is thought to result from the combined action of several proteolytic enzyme systems, including the matrix metalloproteinases (MMPs), serine proteinases, and cysteine proteinases. The majority of the s ...

The essential role of genes is in encoding structural proteins and enzymes which enable the cell or organism to maintain homeostasis in the face of the environmental challenges experienced (1). DNA containing such genetic information varies from one species to another. Even within a speci ...

Mammalian collagenases cleave all three polypeptide chains of the triple helical collagen molecule at a specific site to give characteristic one-quarter and three-quarter fragments. These denature at 37�C becoming susceptible to digestion by less specific proteinases.

Zymography and reverse zymography are techniques used to analyze the activities of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in complex biological samples. The two methods are technically similar. Zymography involves the electr ...

The techniques of Western blotting and immunocytochemistry are suitable for the quantification and localization, respectively, of matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP) expression. However, they do not indicate the endogenous ba ...

How do cells degrade their surrounding matrix? Constitutive and induced cellular secretion of several classes of proteinases have been implicated in extracellular degradation (1) and many of these proteinases have the ability to degrade extracellular matrix proteins in vitro. Ho ...

The matrix metalloproteinases (MMPs) have a pivotal role in both normal and pathological turnover of the extracellular matrix. Whereas MMP protein can easily be detected by immunolocalization or Western blot analysis, the determination of whether or not an MMP is active and acting on a part ...

Collagen is the most abundant protein in the mammalian body. Collagen types I, II, and III are the major structural components of skin, bone, cartilage, and connective tissues. They exist as fibrils of crosslinked helical molecules composed of three α chains of approx 1000 amino acids each, with non ...