细胞功能测定

Changes in cell behavior are driven by changes in gene expression. Thus, in order to understand the mechanisms regulating cell behavior, one has to identify and characterize differentially expressed genes. Standard methods currently used to isolate differentially expressed genes ...

The purification of good-quality RNA from tissue-cultured cells is essential for many applications and several methods exist for the isolation of total RNA. Most protocols rely on sodium dodecyl sulfate (SDS) (1) or guamdium thiocyanate (2,3) to simultaneously lyse cells and mactivate e ...

From the very beginning of PCR technology (1– 3), it was clear that the power of amplification could be used for the study of mRNA expression (4,5). This technique is now wldeiy used and many different protocols have been developed using either viral reverse transcriptases (RT) or exploiting the reve ...

Nonradioactively labeled probes offer several advantages compared to radioactive ones, as they show long stability, high morphologrcal resolution, and rapid developing time. There are different types of nonradioactive labeling methods available, although dlgoxigenin- ...

Methods allowing sensitive and accurate quantitative analysis of defined RNA species are required in a wide variety of gene expression studies. Unlike the traditional hybridization methods, RNase protection or S1 nuclease assays (see Chapters 16 and 27), the methods based on reverse tr ...

As already reported in a previous chapter of this book, it is possible to extract and analyze RNA from fixed- and paraffin-embedded tissues (see Chapter 5). For many purposes, a simple qualitative analysis of the presence of a specific RNA in tissues may be sufficient, for instance, to establish the per ...

Adenosine 3′,5′-cyclic monophosphate (cAMP) is involved in a myriad of normal and pathological processes. Indeed, this cyclic nucleotide serves as a second messenger for the action of endogenous and exogenous agents in organisms ranging from bacteria to humans (1). The ubiquitous nature ...

The generation of cyclic AMP (cAMP) is probably the most scrutinized of signal transduction pathways. Receptors may be linked to the generation of AMP by one of two routes. One group of receptors (such as β-adrenoceptors, A2 adenosine receptors, D1 dopamine receptors, histamine H2 receptors, a ...

In mammals, adenylyl cyclase is a family of membrane-bound enzymes that catalyze the conversion of adenosine triphosphate (ATP) to adenosine 3′:5′-cyclic monophosphate (cAMP). cAMP is an ubiquitous intracellular signaling molecule that modifies cell function by activating cAMP ...

The methods described in this chapter are designed to measure the hydrolysis of guanosine triphosphate (GTP) to inorganic phosphate (Pi) and guanosine diphosphate (GDP), a reaction catalyzed by the GTPase enzymes (EC 3.6.1.-). The theory behind the experimental design involves using GTP as a ...

Nitric oxide (NO) is an important biological mediator involved in control of blood vessel tone, neurotransmission, and immune function (1–3). NO has physicochemical properties that make it ideal to function as an intercell communicator, and these include an ability to travel rapidly bet ...

Phospholipases A2 (PLA2s) are important enzymes in signal transduction, being responsible for release of arachidonate from membrane phospholipids for the production of prostaglandins, leukotrienes, platelet-activating factors, and other bioactive lipids (1). Phospho ...

There is now increasing evidence that phospholipase D (PLD) activity can be stimulated by a range of hormones, growth factors, and neurotransmitters in a range of cell types (1). The enzyme generally catalyzes the hydrolysis of phosphatidylcholine (PtdCho) to produce phosphatidate (Pt ...

The serine/threonine kinase protein kinase C (PKC) is regarded as playing a key role in stimulation of cellular responses in many different cells and tissues. There are now known to be at least 10 different isoenzymes of PKC (α, βI, βII, γ, δ, ɛ,ζ, η, θ, and λ @#@) which vary in their cofactor requirement for activati ...

The enzyme Ca2+/calmodulin-stimulated protein kinase II (CaM-PK II) belongs to a family of calmodulin-stimulated serine/threonine protein kinases that are widely distributed in nature, but are especially enriched in the brain (1–3). CaM-PK II is also found in a number of subcellular frac ...

Many cell-surface receptors via G-proteins activate phosphoinositidase C, which catalyzes the hydrolysis of phosphatidylinositol 4,5-bis-phosphate to produce the second messengers, myo-inositol 1,4,5-trisphosphate and diacylglycerol (1). Ins(1,4,5)P3 interacts w ...

The early methods for calcium measurement involved microinjection of calcium-sensitive proteins, such as aequorin or obelin, into large cells (1, 2) or the use of microelectrodes (3). Both techniques are still employed, however, with much improved sensitivity allowing investigati ...

Diacylglycerol (DAG) found within cells is derived from two broad sources. First, in the de novo biosynthetic pathway, glycerol 3-phosphate is converted by two acylation steps to phosphatidic acid (PA), which is converted to DAG by phosphatidate phosphohydrolase (PPH). This is part of the pa ...

Phosphoinositide hydrolysis by phospholipase C (PLC) is a widespread transduction mechanism by which activation of many neurotransmitter and hormone receptors triggers the formation of the second messengers inositol 1,4,5-trisphosphate and 1,2-diacyl-glycerol (1–3). Co ...

Excitatory amino acid receptors are the predominant type of neurotransmitter receptor in the vertebrate central nervous system (CNS). They are membrane-spanning proteins that mediate the stimulatory actions of glutamate and possibly other related endogenous amino acids. Exc ...