Methods in molecular biology
-
Astrocytes produce numerous mediators under conditions of inflammation in the central nervous system. One such mediator is nitric oxide (NO) derived from nitric oxide synthase-2 (NOS-2), the high output, inducible NOS isoform. Expression of NOS-2 and production of NO can be stimulated in astrocyte cultures by combinations of cytokines and lipopolysaccharide, a gram-negative bacterial endotoxin. This chapter details methods to induce and analyze NOS-2 expression and NO production in astrocyte cultures.
-
For decades, researchers have used animal self-administration models to examine the effects drugs of abuse have on physiology and behavior. Sophisticated self-administration procedures have been developed to model many different aspects of drug addiction. ⋯ In this way, animals can hold the lever down for any duration of time thereby self-administering any dose on a continuous spectrum. This procedure eliminates some of the ambiguity in translating results from effects only observed at one unit dose and allows examination of which dose the animal "prefers" at different times.
-
Osteoarthritis (OA) is one form of degenerative joint disease characterized by progressive loss of articular cartilage, decreased function and is frequently accompanied by chronic pain. Given the success of arthroplasty as a treatment for late-stage OA, there is considerable interest in developing therapies pertaining to the management of pain associated with OA as well as therapies designed to slow or reverse the progression of the disease. ⋯ Here, we describe a model of OA-related pain in which progressive joint destruction is induced by injection of monosodium iodoacetate into the articular space of the knee of the rat. Further, we describe three different methods to measure pain-related behaviors in this model: hind limb weight bearing, primary mechanical hyperalgesia, and hind limb grip strength.
-
G-quadruplexes are noncanonical secondary structures formed in DNA sequences containing consecutive runs of guanines. DNA G-quadruplexes have recently emerged as attractive cancer therapeutic targets. It has been shown that the 3' G-rich single-stranded overhangs of human telomeres can form G-quadruplex structures. ⋯ Nuclear magnetic resonance (NMR) spectroscopy has been shown to be a powerful method in determining the G-quadruplex structures under physiologically relevant conditions. We present the NMR methodology used in our research group for structure determination of G-quadruplexes in solution and their interactions with small molecule compounds. An example of a G-quadruplex structure formed in the human telomere sequence recently solved in our laboratory is used as an example.
-
Large numbers of diverse small non-coding RNAs have been discovered and characterized in eukaryotic RNA interference pathways. These small RNAs have distinctive characteristics and are associated with Argonaute family proteins to regulate gene expression and genomes at various levels. These small RNAs include the Dicer-dependent group such as microRNAs (miRNAs) and small interfering RNAs (siRNAs), and the Dicer-independent group such as Piwi-interacting RNAs (piRNAs). This review summarizes the various classes of eukaryotic small RNAs and the general knowledge of their characteristics, biogenesis, and functions, with emphasis on some of the recently identified small RNAs.