实验仪器使用技术

The typical mammalian genome contains 3 � lo9bp of DNA. This amount of DNA is believed to include 3-4 � lo4 unique protein- encoding genes. Even complex, highly specialized cells, such as neurons, express a small fraction, perhaps 20%, of all the possible genes, how- ever. A major problem in neurobiology, th ...

Recent progress in the field of molecular biology has provided a new method to localize an mRNA signal within a cell or tissue section. This technique, called “in situ hybridization histochemistry,” gives us dynamic information about gene expression at individual cellular lev- els. For the de ...

Because of the vast organizational complexity of the brain, it is crucial to study gene regulation at the level of individual cells. Most brain regions or individual neurons lying adjacent to each other may contain different neurotransmitters or different receptors, which may differen ...

Human mitochondrial DNA (mtDNA) is a closed circular genome of 16,569 bp (1), encoding 13 subunits of four enzyme complexes (Complexes I, III, IV, and V) in the oxidative phosphorylation system (2). Mutations of mtDNA have been demonstrated to be associated with various neuromuscular diseases. ...

Chemically synthesized oligonucleotides are powerful tools in the molecular biologist’s repertoire. This chapter describes the design of oligonucleotides and their applications, with particular reference to their use in the isolation and characterization of recombina ...

The polymerase chain reaction (PCR; 1) employs a pair of oligonucleotide primers, one sense and one antisense, with respect to the template DNA. During repeated cycles of heat denaturation, annealing of the oligonucleotide primers to the template and extension of the primers by the enzyme taq p ...

All cells have a large number of proteins; for instance, a single hepatocyte has about lo4 different proteins, and a number of them are involved in tissue-specific functions. In neuronal cells, there are additional 104 protein species, which are predicted, from kinetic of DNA-RNA hybridizati ...

The complexity of neural gene expression results from an inter- play of a multitude of signal transduction pathways and trans-acting transcription factors interacting at cis-acting DNA elements (1). Clearly, a major challenge is to define the pathways and molecular mechanism(s) inter ...

The appearance and maintenance of differentiated neural cell types in eukaryotic organisms can be largely attributed to characteristic spa- tial and temporal regulatory cascades evoking cell-specific gene expres- sion. Since gene expression causes information passage from D ...

This chapter describes procedures for purification of μ-calpain, m-calpain, and calpastatin simultaneously from a single tissue sample. The procedures and reagents described apply equally well to purification of any one or two of these three proteins from an appropriate tissue with a c ...

Although in recent years the molecular structure and function of calpains have been extensively investigated, some aspects of the roles and properties of these proteinases remain obscure, particularly their precise function in the cell, and the mechanisms inducing and promoting th ...

Determination of calpastatin levels in cells or tissues by Western blotting using antibodies, or by assay of calpain inhibitory activities in vitro, may be required to assess the role and the level of calpastatin, with respect to the levels of other components of the calpain system, in certain cell ...

Calpastatin is an endogenous inhibitor protein specific for calpain (1,2). Other calpain-inhibiting reagents such as EDTA, E-64, leupeptin, and calpain inhibitor I (N-acetyl-L-leucyl-L-leucyl-L-norleucinal), are frequently used in attempts to assess the role of calpain in degra ...

In many cases separation of calpains and calpastatin has to be performed with a relatively small amount of tissue, so that large-scale and complicated multiple-column procedures are not appropriate. In designing a method suitable for small amounts of tissue, it is important that the extrac ...

The methods described in this chapter are designed for rapid and convenient separation and quantitation of calpain isozymes in cultured osteoblastic cells by modern semiautomated strong-anion exchange FPLC. The isozymes of the calpain-calpastatin system are abundant in osteo ...

There is no single affinity chromatography method that is universally effective for the purification of calpains. Dye affinity chromatography and calcium-dependent binding to immobilized substrates or inhibitors are the most commonly attempted affinity methods. Our labor ...

The purification of μ-and m-calpain on a medium to large scale from animal tissues such as bovine heart or skeletal muscle, and on a microscale from less than 0.5 g of tissue, is described in Chapters 1 and 2. These preparations are excellent for many purposes, but for some other purposes, for example for cryst ...

Calcium-dependent cysteine proteinases (CDPs or calpain-like proteinases) constitute a large family of related proteins in the tissues of invertebrate species. They vary in native mass from 59 kDa for lobster muscle CDP III to 520 kDa for octopus muscle CDP, and vary also in subunit composit ...

p94 (also called calpain 3, nCL-1, or CAPN3) is a calpain large subunit homologue, which is predominantly expressed in skeletal muscle (1–4). The mRNA level of p94 in skeletal muscle is at least 10 times higher than that for the conventional calpain subunits (1). However, the p94 protein is not easily dete ...

Methods for analyzing enzyme activity after electrophoresis in acrylamide gels have been described for a variety of proteins, including several proteases and kinases. The application of this approach to detect calpain activity by casein zymography was first described by Raser et al. in ...