实验仪器使用技术

Peptides are very flexible molecules, in contrast to proteins, which are stabilized by disulfide bridges and salt bridges in their tertiary structure. Peptides can adopt several conformations, at least in aqueous solutions. Some limitations, however, are imposed caused by then prima ...

Size-exclusion high performance liquid chromatography (HPLC) has enabled purification of peptides and proteins to be carried out 10–100 times faster than conventional gel filtration chromatography on soft gels. The prepacked columns (about 1 � 30 cm) contain small particles (13 �m or l ...

Synthesis of peptides on a solid support is described in detail in Chapter 5 of this book. Contributing to the ongoing success of Merrifield’s solid-phase peptide synthesis methodology (1) was the use of high performance liquid chromatography (HPLC) for the purification of the desired pept ...

This protocol combines in situ hybridization techniques with immunocytochemistry (ICC). Such a protocol generally allows you to identify cells that have both a specific species of mRNA expressed and a separate specific protein. This combination of techniques is particularly powe ...

A large variety of different neuropeptides function as central transmitters in neuronal circuits regulating diverse aspects of energy homeostasis such as feeding behavior, energy expenditure, and thermoregulation. Neuropeptide Y (NPY) represents one such peptide mediati ...

In situ hybridization histochemistry (ISHH), first described in 1969 (1,2), allows a specific complementary RNA species to be detected directly at its site of expression, revealing its cellular localization and relative abundance. The utilization as a label of digoxigenin (3), which is n ...

Antisense oligodeoxynucleotides have been successfully employed to study the function of viral and cellular genes by sequence-specific inhibition. The antisense oligodeoxynucleotides are single-stranded, short, native, or chemically modified nucleotides that inhi ...

Antisense oligodeoxynucleotides (ODN) that disrupt gene function, leading eventually to a reduction in levels of the encoded protein, are powerful molecular tools for studying the functions of neuropeptides and their receptors in the brain. Their potential usefulness is even grea ...

Neuropeptide Y (NPY), peptide YY (PYY), and pancreatic polypeptide (PP) are endogenous 36-amino acid peptides belonging to the same family. Different receptor subtypes have been identified in the NPY/PYY/PP family (1), and mammalian subtypes are now classified collectively as NPY rece ...

Following its discovery in rat intestinal epithelial cells (1), the peptide YY (PYY) receptor has been characterized in the proximal tubule PKSV-PCT cell line derived from kidneys of SV40 large T antigen transgenic mice (2). This receptor is PYY preferring since it binds the intestinal hormone ...

Energy homeostasis is the process by which adipose tissue, the stored body energy, is kept constant over time. Obviously, feeding behavior plays a crucial role in energy homeostasis. Therefore, to maintain a constant energy balance, energy (food) intake should match energy expenditure. A n ...

Transgenic technology provides a powerful tool to study the function of a novel gene in the whole organism. The technology of transgenic rat production is receiving increasing attention. The rat is the model of choice in cardiovascular, endocrine, behavioral, and toxicology studies. This ...

Cloning strategies based on homology provide an attractive methodology for identifying novel members of a multigene family. The techniques are straightforward, with well-established protocols, and high-quality reagents are readily available. Most steps are relatively for ...

Neuropeptide Y (NPY) is one of the most abundant neuropeptides in the mammalian nervous system and is an important mediator of numerous physiological effects (1). NPY elicits its manifold biological effects by interacting with at least six different G-protein-coupled receptors known ...

The Y4-receptor subtype shows high species diversity compared with the Y1-, Y2-, and Y5-receptor subtypes. The rodent Y4 receptor differs considerably in sequence, pharmacology, and distribution from the human Y4 receptor. To characterize further the intriguing species differenc ...

Our interest in G-protein signaling events involved in the specialized functions of the retinal pigment epithelium led us to investigate the pattern of G-protein-coupled receptor expression in this cell type. We used reverse transcriptase-coupled polymerase chain reaction (PCR) ...

The methods used to isolate genomic clones of a particular gene have changed significantly over the years. In particular, the characterization of genes with many exons, separated by large introns, increased the need for clones with large inserts. The average length of an insert contained wit ...

The nervous system is composed of a diverse population of cells selectively expressing different genes which ultimately determine the type of neurotransmitters and neuropeptides produced and released. Neuropeptide tyrosine (NPY) is one of the most abundant and widely expressed p ...

In studies on pluripotency and differentiation, teratocarcinoma cell lines have been used as alternatives to mouse embryonic stem cell lines. Teratocarcinoma cell lines cost less to maintain and they are easier to genetically manipulate. Their disadvantage, of course, is being deri ...

Signal transduction is a key process to transmit information from the extracellular milieu, and to elicit changes in the biological activity of target cells. Several cell signaling pathways can be targeted by neurotoxicants and developmental neurotoxicants. This chapter focuses ...