生物化学技术

In recent years, molecular tools have been developed to identify and quantify specific microbial cells, or specific microbial activittes, in mixed populations (1, 2). These tools are especially valuable for analysis of specific bacteria in complex environmental samples. Increasi ...

Peroxynitrite (ONOO−) is formed by the reaction of nitric oxide (NO) with superoxide (O2 −) (1). Under normal circumstances, these two free radicals are not formed at similar rates in the same cellular or extracellular compartment; however, under some pathological conditions, such as inflam ...

Biologically produced nitric oxide (NO) originates from oxygen and L-arginine in the reaction catalyzed by NO synthase (NOS) enzymes (NOS, EC 1.14.13.39), first reported in the late 1980s. It was soon discovered that these enzymes have different biochemical properties depending on the tis ...

The endogenous production of nitric oxide (NO) plays a key role in many bioregulatory systems, including the control of vascular tone, inhibition of platelet aggregation, neurotransmission, and macrophage toxicity. Many of these pathways are activated by the stimulation of soluble g ...

In-situ hybridization (ISH) is a technique that allows the demonstration of nucleic-acid sequences of interest within tissues and their localization to subpopulations of cells within those tissues.

Three distinct isoforms of nitric oxide synthases (NOS) have so far been identified and cloned (1). There are two constitutively expressed NOS—neuronal (n) and endothelial (e), NOS—and one inducible (i) form, iNOS. All three have been demonstrated immunochemically using antibodies rai ...

The amount of nitric oxide synthase (NOS) in tissues and cells can readily be determined by Western blotting. Antibodies are now commercially available for the detection of the inducible (i), neuronal (n), and endothelial (e) NOS. These antibodies are specific for the particular isoforms of NOS ...

The amount of RNA varies greatly between different cell types and organisms, and the majority of RNA (more than 95%) consists of ribosomal RNA (28S, 18S, and 5S species) and the tRNA. Only a small proportion (usually less than 1% of the total amount) of RNA corresponds to the mRNA species. However, in order to imp ...

Numerous cell populations are capable of producing nitric oxide (NO) from L-arginine. The nitric oxide synthases (NOS) catalyzing the formation of NO have been grouped into two broad categories: the constitutive Ca2+/calmodulin (CaM)-dependent endothelial NOS (eNOS) and neuronal N ...

Absorption spectroscopy is based upon the principle that the absorption of radiation by a molecule involves the transition of the energy level from its ground state to an excited state. If we denote the energy difference as ΔE and the frequency of radiation as ν, then the relationship is expressed as ( ...

With an extremely short half life and susceptibility to postsynthetic oxidation, it has been a challenge to measure nitric oxide (NO). This chapter discusses the chemiluminescence assay for NO and its intermediate and end products of oxidation—NO2 − and NO3 −. This is one of the most simple, sensit ...

Reliable methods for the specific detection and quantitative determination of nitric oxide (NO) release from tissues are a prerequisite for a better understanding of the complex biological functions of this widespread cellular messenger. Although several techniques are avail ...

Nitric oxide (NO) produced from L-arginine during the activation of nitric oxide synthase(s) (NOS) in cells has pleiotropic effects and can behave as an intracellular modulator of cellular activity, as well as an intercellular messenger which diffuses from the originator cell to affect ot ...

This chapter reviews the CE analysis of bases, nucleosides, and single-stranded (oligo)nucleotides. The analysis of double-stranded DNA and the technology associated with capillary gel electrophoresis (CGE) is covered elsewhere in this book (Chapter 13) and in recent reviews (1–3). A ...

The majority of drugs are either acidic or basic water-soluble compounds, and many drugs may be adequately separated by FSCE. For example, an acidic drug may be analyzed in its anionic form at high pH, and basic drugs may be tested at low pH in their cationic form. Figure 1 shows the efficient separation of a test m ...

Peptide analysis is routinely performed using reversed-phase liquid chromatography (RP-HPLC), which achieves separation based on hydrophobicity differences between peptides (1,2). Recently, however, capillary electrophoresis (CE) has increasingly been used for pept ...

A variety of free solution CE (FSCE) methods have been developed for the determination of low-mol-wt solutes. These samples have included metal ions, small amine compounds, small organic acids, and a range of inorganic anions. Separation mechanisms involve either simple FSCE or addition of a s ...

The T7 transcription method from single-stranded templates with a double-stranded promoter is a common method of preparing RNA for in vitro ribozyme studies (1). Although the identification of full-sized ribozyme transcripts IS fairly straightforward after gel separation, the sa ...

This chapter provides the basic procedures for RNA handling, transcription reactions, and transcript purification (1,2). For further analysis, the RNAs can be end-labeled. Simple procedures generate a sequence ladder to confirm colinearity of template and transcript (3,4). This is n ...

This chapter provides protocols for two different methods that require modified transcripts, modification interference and phosphorothioate footprinting. Modification interference is used to identify positions (in substrate or ribozyme RNAs) where modified nucleo ...