生物化学技术

Synthesis of modified nucleotides and then incorporation into an RNA chain have facilitated our understanding of the functron and mode of action of specific nucleotides within the ribonucleic acid. Recent developments in the chemical and enzymatic synthesis of RNAs (1–3) have allowed ...

Catalytic antisense RNAs combine two RNA-based strategies for gene suppression: the antisense approach and the ribozyme approach. Like ordinary antisense RNA, catalytic antisense RNAs are characterized by a relatively long stretch of sequences (30 bases) that are complementary ...

With the advent of antisense technology, there has been much interest in the use of long RNAs expressed in vivo to inhibit the expression of target genes. More recently, there have been numerous reports that the incorporation of either hairpin or hammerhead ribozyme motifs (catalytic antise ...

Asymmetric hammerhead ribozymes represent a variation of catalytic antisense RNAs that have been introduced in this book as a specialized form of ribozymes in Chapter 14. Catalytic antisense RNAs are characterized by a relatively long antisense sequence (30 bases) that allow efficie ...

The hammerhead ribozyme as engineered by Haseloff and Gerlach (1) consists of the conserved nucleotides C3-A9 and G12-C15.2 connected by nucleotides that form stem-loop II, and nucleotides at the 5′- and 3′-ends, which form helix I and helix III, respectively, in complex with the substrate. Such a ri ...

Following the discovery of catalytic RNA (1), a number of different ribozymes have been found. Most ribozymes carry out site-specific cleavage of the RNA phosphodiester backbone, although important exceptions may be emerging (2,3). The catalytic center and reaction site of several natu ...

Transmembrane receptors play a crucial role in communication between cells. By coupling to intracellular proteins and second-messenger systems, these molecular entities allow for the transduction of a biochemical message across the cell membrane. Early investigations of rec ...

The development of new and improved analytical methods for the study of eicosanoid biosynthesis has enabled researchers to identify and profile products that are generated by individual cell types in vitro. In vivo scenarios encountered in inflammation, thrombosis, atheroscler ...

Arachidonic acid is a polyunsaturated fatty acid that is esterified at the sn-2 position of the glycerol backbone of membrane phospholipids. Hormonal and paracrine stimuli can activate phospholipases that cleave arachidonic acid from membrane phospholipids. The free intracel ...

Prostaglandin synthases are key enzymes in the prostanoid biosynthetic pathway. Arachidonic acid released from membrane phospholipid stores by the activity of phospholipases is converted to PGH2, a common precursor of prostaglandins, by the enzymatic activity of prostaglan ...

5-Lipoxygenase (5-LO) catalyzes the conversion of arachidonic acid to leukotriene mediators of inflammation (1,2). Leukotriene biosynthesis in intact cells involves redistribution of 5-LO from a soluble cellular compartment where it is inactive to the nuclear envelope where its ...

Extravasation of neutrophils is an early and critical event during an acute inflammatory response. The process of emigration of leukocytes through the wall of an intact capillary has been described as a “flowing” between endothelial cells (1); i.e., the leukocyte reaches the perivascular ...

Researchers in the eicosanoid field have frequently applied whole blood assays (1–3) for evaluation of enzymic inhibitors in preclinical and clinical studies. In numerous investigations of antiplatelet agents, the capacity of blood to generate thromboxane A2 (TXA2) in response to an ...

Expression of foreign proteins in both cultured and intact mammalian cells has become essential to evaluating cellular biology and physiology including the proteins involved in the control of eicosanoid production. Many transient expression vectors are commercially availa ...

This chapter will summarize the biochemistry and biological actions of isoprostanes (IsoPs) and related compounds. Several comprehensive reviews of the IsoPs have been published recently (1–3). Therefore, many aspects of this subject will only be summarized briefly for sake of compl ...

Many analytical methods for eicosanoids require extraction of these substances from biological fluids or media prior to analysis, and even when not essential, extraction can be used to increase both the specificity and sensitivity of assays. Extraction and chromatography of eicosa ...

The acid guanidinium-thiocyanate phenol chloroform (AGPC) procedure is the principal method used for one-step RNA extraction and is a predominant choice for isolation of total RNA from a wide variety of biological samples. (Note 1) (1,2). This procedure permits not only isolation of RNA from l ...

Metabolism of arachidonic acid results in formation of eicosanoids including prostaglandins (PGs), thromboxanes (Txs), monohydroxyeicosatetraenocinoids (HETEs), leukotrienes (LTs), lipoxins (Lxs), epoxy metabolites, and hepoxilins (1). Most of these compounds are fo ...

Immunocytochemical analysis is an effective method for providing a differential assessment of protein expression and localization in tissue samples. A variety of methods exist for immunocytochemical analysis including: immunofluorescence, immunoperoxidase, and str ...

Prostaglandin production is dependent on the presence of an enzyme called prostaglandin G/H synthase, better known as cyclooxygenase, that catalyzes the first steps in the conversion from arachidonic acid. A cDNA for cyclooxygenase was first cloned from sheep seminal vesicles in 1988 ( ...