生物化学技术

Synthetic oligodeoxyribonucleotides (ODN) have been proposed as a class of potential therapeutic agents that can interact in a rational way with DNA or RNA, with the aim of inhibiting the expression of unwanted genetic information (1–3). One of the most critical questions in the evaluation of ...

RNA molecules contain a number of modified nucleosides, in addition to the normal ribonucleosides adenosine, guanosine, cytidine, and uridine. More than 90 different modified nucleosides with great structural diversity have been described in RNA (1), most of them in tRNA, but some also in rR ...

As discussed in Chapter 4, exoglycosidases are useful reagents for the structural determination of glycoconjugates. Their anomeric, residue, and linkage specificity for terminal monosaccharides have been used to assess monosaccharide sequence and structure in a variety of gly ...

The purification of oligosaccharides and glycopeptides presents many problems, not the least of which is the diversity of possible structures. To date no single chromatographic or electrophoretic method has been proven to separate all oligosaccharide isomers in a buffer system that ...

The pyridylamination method was originally described in 1978 as a means of analyzing glycan structures with high-sensitivity (1). Subsequently, the method has been applied to structure analyses of glycans including glycosidase digestion (2), 2D-mapping by various kinds of high-pe ...

The biosynthesis of glycosaminoglycans (GAGs) and proteoglycans appears to be a ubiquitous function in mammalian cells. Some biological sources, notably connective tissue, produce large quantities of GAGs that can be readily detected by colorimetric assays and, therefore, may be i ...

Prior to 1980, most methods for analysis of glycoprotein carbohydrates utilized column, thin-layer, and paper chromatography, gas chromatography, mass spectroscopy, and rarely nuclear magnetic resonance spectroscopy. These methods required relatively large amounts of ma ...

This chapter will attempt to provide an overview of the theory and details of some basic applications of the technique of confocal microscopy. Since this is intended to be a general introduction to the possibilities available, it is not appropriate to include detailed descriptions of the spec ...

Intracellular calcium, in particular the cytosolic free ionized calcium concentration i, is tightly regulated under physiological conditions. Stimulation of receptors, belonging to almost all the classes so far described, will result in changes in i. These changes might be directly ...

Many cellular functions are tightly regulated by intracellular calcium concentrations (i), and, therefore, the measurement of i is of critical importance. To determine Ca2+-related cellular dynamics accurately, it is necessary to measure three-dimensionally resolved i with su ...

Ionized calcium plays a major role in the regulation of cellular processes in both eukaryotic and prokaryotic cells (1). A wide variety of cell surface receptors and ion channels utilize a calcium signal to initiate events such as cell motility, contraction, and secretion. To a large extent, the ad ...

For decades, the measurement of extracellular Ca2+(40Cao 2+) entry into cells, using 45Ca2+ as carrier, has been a widely used technique. One of the first studies was performed by Hodgkin and Keynes (1) to measure the rate at which 40Ca2+ labeled with 45Ca2+ crosses the surface membrane of resting squid a ...

Intracellular Ca2+ (i) can rise primarily via release from intracellular storage sites (e.g., the endoplasmic reticulum) or via entry across the membrane down the steep concentration gradient (1–3). Ca2+ can enter through two main classes of plasma membrane-located Ca2+ channels: recep ...

Hormones, neurotransmitters, chemoattractants, and growth factors all elicit intracellular responses, on binding to cell surface receptors, by activating inositol phospholipid-specific phospholipase C (PLC). Activated PLC catalyzes the hydrolysis of phosphatidyl ...

Many cell surface receptors activate phosphoinositidase(s) C, via G proteins that catalyze the hydrolysis of phosphatidylinositol 4,5-biphosphate to produce the second messengers, inositol(1,4,5)trisphosphate and diacylglycerol (1). Ins(1,4,5)P3 interacts with speci ...

Many receptor tyrosine kinases and seven-transmembrane receptors are directly coupled or coupled via G proteins, respectively, to the activation of phosphoinositidase Cs (1). These enzymes catalyze the hydrolysis of phosphatidylinositol 4,5-bisphosphate to produce the se ...

Photolabile calcium chelators have been designed to study intracellular calcium-dependent processes (1,2). These “caged” calcium compounds are useful tools to bypass rate limiting steps, i.e., in muscular excitation-contraction coupling (3–5). When using chelators intrace ...

The inositol (1,4,5)trisphosphate receptor is an intracellular Ca2+ release channel located within the membrane of the smooth endoplasmic reticulum (ER). The study of kinetics and regulation of the Ins(1,4,5)P3 receptor is greatly complicated by problems of the heterogeneity of its en ...

Inositol(1,4,5)trisphosphate is a well-characterized second messenger that interacts specifically with a family of Ins(1,4,5)P3 receptor-operated Ca2+ channels to mobilize nonmitochondrial intracellular Ca2+ stores (1). Ins(1,4,5)P3-induced Ca2+ mobilization (IICM) ...

The fact that acetoxymethyl (AM)-ester derivatives of fluorescent Ca2+ indicators accumulate not only in the cytoplasm but also in organelles was recognized long ago as a potential source of artifacts during measurements of cytoplasmic (1,2). Later it was observed that high-affinity d ...