细胞功能测定

Quantitative studies are increasingly found in the literature, particularly in the fields of development/evolution, pathology, and neurosciences. Image digitalization converts tissue images into a numeric form by dividing them into very small regions termed picture elemen ...

Neoepitope antibodies recognize the newly created N or C terminus of protein degradation products but fail to recognize the same sequence of amino acids present in intact or undigested protein. Aggrecan neoepitope antibodies have been pivotal in studies determining the contributi ...

The exuberant expression of proteinases by tumor cells has long been associated with the breakdown of the extracellular matrix, tumor invasion, and metastasis to distant organs. There are both epidemiological and experimental data that support a causative role for proteinases of the m ...

Zymography is the electrophoretic separation of proteins through a polyacrylamide gel containing a proteolytic substrate. After denaturing (but nonreducing) electrophoresis, proteins are renatured and incubated in an appropriate buffer for proteolytic activity. Clear ...

Quantitative reverse transcriptase polymerase chain reaction enables the accurate quantification of gene expression in cultured cells or small tissue samples. In this chapter, we describe the use of Taqman� technology to measure expression of matrix metalloproteinases and re ...

Many molecular genetic studies of human diseases involve determining the genotypes for single nucleotide polymorphisms. This chapter summarises a number of different techniques for the single nucleotide polymorphism genotyping which can be applied to MMP genes. The chapter also ...

The degradome microarray – CLIP-CHIP™ – is a dedicated and focused array that allows the analysis of all proteases, non-proteolytic homologs, and protease inhibitor gene transcripts in the human and murine genomes at the mRNA transcript level. Based on unique 70-mer oligonucleotides, des ...

This 14-day model of cartilage breakdown involves stimulation of bovine nasal cartilage with a combination of interleukin-1 and oncostatin M. Media is harvested on days 7 and 14 and the conditioned media and remaining cartilage at day 14 assayed for the levels of proteoglycan and collagen fra ...

This chapter describes the production and characterization of antibodies raised against neoepitopes in collagenase-cleaved collagen. It also details the development, validation, and use of immunoassays using such antibodies to measure specifically collagenase-medi ...

Analysing cell migration and invasion is of interest to many investigators as they mimic a part of physiological or pathological events. In this chapter, methods to analyse MMP-dependent 2D and 3D cell migration are described in detail. To study 2D cell migration, the phagokinetic track moti ...

Tissue inhibitors of metalloproteinases (TIMPs) are a group of highly potent inhibitors of matrix metalloproteinases (MMPs) and disintegrin metalloproteinases (ADAMs). The high affinity and “tight-binding” nature of the inhibition of MMPs or ADAMs by TIMPs presents challenges ...

Identification of protease substrates is essential to understand the functional consequences of normal proteolytic processing and dysregulated proteolysis in disease. Quantitative proteomics and mass spectrometry can be used to identify protease substrates in the cell ...

The recognition that the successful clinical use of MMP inhibitors will require quantitative correlation of MMP activity with disease type, and to disease progression, has stimulated intensive effort toward the development of sensitive assay methods, improved analytical meth ...

Metalloproteases comprise a heterogeneous group of proteolytic enzymes whose main characteristic is the utilization of a metal ion to polarize a water molecule and perform hydrolytic reactions. These enzymes represent the most densely populated catalytic class of proteases in m ...

The expression of a recombinant MMP in a mammalian cell line can be useful, e.g., for purification of the enzyme, to characterize function of the enzyme, or to uncover its substrates. In this chapter, we have therefore documented our experience with the recently discovered MMP-28.

As their name implies, matrix metalloproteinases (MMPs) are thought to be responsible for the turnover of connective tissue proteins, a function that is indeed performed by some family members. However, matrix degradation is possibly not the predominant function of these enzymes. Seve ...

The “a disintegrin and metalloproteinase with thrombospondin motifs” (ADAMTS) enzymes are secreted proteinases involved in development, blood clotting and the turnover of extracellular matrix. Manufacturing recombinant enzyme presents quite a challenge due to the presen ...

Matrix metalloproteinases (MMPs) modulate cell–cell and cell–matrix interactions and the membrane-type MMPs (MT-MMPs) are especially important for pericellular proteolysis. Understanding the activity, regulation, and function of MT-MMPs would provide important insi ...

Signal Transducer and Activator of Transcription (STAT) proteins are latent cytoplasmic transcription �factors that become activated by phosphorylation at a C-terminal tyrosine residue. Upon activation STAT proteins translocate to the nucleus and bind to their specific tar ...

JAK kinases are critical mediators in development, differentiation, and homeostasis and accordingly, have become well-validated targets for drug discovery efforts. In recent years, the integration of X-ray crystallography in kinase-focused drug discovery programs has prov ...