Tissue, slices

Slices and other parts of tissues of animal or plant origin are the most complex biosystems so far applied in biosensors. Tissues containing large amounts of the enzymes of interest have been deliberately used. An overview of tissue-based sensors is given in Table 18. 

Since tissue slices are easy to handle, they can be immobilized on electrodes by simple mechanical fixation using a semipermeable membrane or a nylon net. Additional crosslinking has been proposed in order to increase the mechanical stability (Kuriyama and Rechnitz, 1981). 

Tissue (slice) Substrate Species detected References 

Pig kidney L-glutamine nh3 Rechnitz et al. (1979) Arnold and Rechnitz (1980a,b) 

Rabbit muscle adenosine monophosphate nh3 Arnold and Rechnitz (1981) 

The thickness of slices obviously has an impact on the accumulation of ammo acids (Sershen and Lajtha, 1974) Glutamate, aspartate, and GABA are accumulated to a greater extent m thinner (0.1 mm) slices than m thicker (0.42 mm) slices. Glutamate seems to accumulate at the surface of thick slices, and thin slices may be damaged to a greater extent than thick ones (Cohen, 1974). 

The use of tissue slices is a standard technique in all aspects of metabolic biochemistry. The tissue is cut into slices, sufficiently thin to allow adequate rates of diffusion in and out of the tissue. The slices are submerged in physiological saline to which substrates or other compounds may be added. 

Changes in the composition of the slices and/or incubation medium give some indication of metabolic activity, but extensive damage may be caused to the cells on slicing the system is so artificial that data obtained by the tissue slice technique may not pertain to the physiological situation. However, the technique is widely used at least for introductory, exploratory experiments. 

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Noradrenaline release might also be modulated by receptors on noradrenergic nerve terminals that are activated by other neurotransmitters ( heteroceptors ). Unfortunately, most studies of this type of modulation have been carried out in tissue slices and... 

Biocatalytic membrane electrodes have an ISE or a gas sensing electrode in contact with a thin layer of biocatalytic material, which can be an immobilized enzyme, bacterial particles or a tissue slice, as shown in Fig. 3 The biocatalyst converts substrate (the analyte) into product, which is measured by the electrode. Electrodes of this type are often referred to as biosensors . 

The concept of a biocatalytic membrane electrode has been extended to the use of a tissue slice as the catalytic layer. An example of this approach is an electrode for AMP which consists of a slice of rabbit muscle adjacent to an ammonia gas electrode. NHj is produced by enzymatic action of rabbit muscle constituents on AMP The electrode exhibits a linear range of 1.4 x 10 to 1.0 x 10 M with a response time varying from 2.5 to 8.5 min, depending on the concentration. Electrode lifetime is about 28 days when stored between use in buffer with sodium azide to prevent bacterial growth. Excellent selectivity enables AMP to be determined in serum. 

Studies on in vitro systems of various kinds (including whole perfused organs, tissue slices, cell, tissue and organotypic cultures, and sub cellular fractions). 

Phenolic acids and aliphatic acids are both involved in the biosynthesis of suberin, and phenolic acids are not synthesized in tissue slices that do not undergo suberization. 

The time-course of deposition of aromatic monomers into the polymer laid down by suberizing tissue slices indicates that the phenolic matrix is deposited simultaneously with or slightly before the aliphatic components. The specific anionic peroxidase appeared with a time-course consistent with its involvement in the polymerization and deposition of the phenolic matrix of the suberin. Increase or decrease in suberin content involves similar changes in both the aliphatic and aromatic components and such changes are associated with the expected increase or decrease in the anionic peroxidase activity caused by physical or biological stress. 

In IMS, supportive materials, whose surfaces are coated with conductive materials, are used in principal. In the simplest way, the tissue slices can be placed on a metal MALDI plate directly.9 In this case, however, the target plate must be cleaned carefully after the measurement is over. Currently, the method commonly used is that samples are prepared on a disposable plastic sheet or a glass slide coated with series of conductive materials. In particular, a plastic sheet (ITO sheet) or glass slide (ITO glass slide available from Bruker Daltonics K.K., Billerica, MA, or Sigma, St. Louis, MO) coated with ITO (indium-tin oxide) is useful because it has superior optical transparency... 

The biocatalyst is incorporated in different physical forms as an isolated enzyme, a whole cell, a subcellular organelle or a tissue slice depending on... 

Stadie, W.C. and Riggs, B.C. (1944). Microtome for the preparation of tissue slices for metabolic studies of tissues in vitro. J. Biol. Chem. 154 687-690. 

Cause of fluoroacetate poisoning In 1947 Bartlett and Barron,1 using tissue slice, showed that fluoroacetate blocked the oxidation of acetate competitively, and that this accounted for the toxic effect, namely, the body was deprived of acetate. Liebig and Peters then found that fluoroacetate blocked the oxidation of fumarate in a guinea-pig s kidney homogenate without accumulation of acetate hence Bartlett and Barron s hypothesis could not be the whole story. 

Because disrupted tissue preparations were unsatisfactory, attempts were made to work either with more organized systems such as tissue slices (liver-Krebs) or to identify and isolate the intracellular organelles involved in the reactions. Cytochemical procedures were developed in the 1930s and 1940s to locate sites of reaction in situ in cells (Chapter 9). Examination of cell ultrastructure became possible when the electron microscope was introduced after 1945. Techniques for the isolation of cell organelles, notably mitochondria, were developed about this time (Chapter 9). 

Fifty years were to pass before it became possible to define the individual enzyme steps in the 6-oxidative process outlined above. No intermediates in the pathway could be found in vivo nor was it possible to detect them in the isolated systems then in use, such as tissue slices. Simpler preparations were needed before the details of the enzymology could be established. 

Nave R, Fisher R, Zech K (2006) In vitro metabolism of ciclesonide in human lung and liver precision-cut tissue slices. Biopharm Drug Dispos 27 197-207. 

LC/MS/MS with selected reaction monitoring (SRM) offers a fast and simple means to analyze biological matrices, which is a key factor in high-throughput CYP inhibition screens using liver microsomes. Potentially, the LC/MS/MS technique is suitable for analyses of cocktail substrates in other in vitro drug metabolism evaluations such as CYP induction/activation assays, rapid analysis of pooled liver microsomes, rapid reaction phenotyping of tissue (hepatic and extrahepatic) samples, as well as evaluation of hepatocytes/tissue slice CYP activity. ° ... 

It is well established that nicotine, and other nicotinic agonists, will elicit Ca++-dependent release of dopamine from striatal tissue slices (see, for examples... 

Immerse the chuck with the tissue slice into the freezing solution. Freezing is indicated by a change in color of the embedding solution from clear to white and will be completed in 5-10 s. After 10 s, remove the chuck and tissue from the freezing solution, and place them on dry ice (see Note 5). 

When staining is to be performed, place the chuck containing the frozen tissue slice in the cryostat for preparation of frozen sections. 

Experimental samples are mainly derived from tissue culture cells, laboratory animals, or human tissues collected from hospitals after surgical biopsies and autopsies. With human and animal tissue specimens, it is important to arrest metabolic processes within 5-10 min of collection in order to preserve mRNAs from degradation by internal enzymatic reactions (26,27). Most hospitals use 10% buffered formalin as a tissue fixative. Subsequently, each tissue slice is trapped in a paraffin block. Series of 4-5-pm-thick sections are cut and mounted on silanated slides. Formalin-fixed archival tissues have been successfully used in in situ PCR and in situ hybridization protocols (28-32). However, the procedure for RNA protection is not always followed. It is often difficult to alter or control the routine procedures of hospitals for the required protection of mRNAs in surgically removed human tissues. 

Another important application of cDNAs is to identify specific proteins in a tissue homogenate or tissue section. Since cDNAs undergo complementary base pairing, adding a radioactively labelled cDNA to a homogenate or tissue slice will bind it to the complementary sequence by a process of hybridization. Thus the amount of radioactive cDNA that hybridizes to the tissue or tissue extract is a measure of the amount of mRNA that is complementary to it. When this procedure is undertaken on slices of brain, it is known as in situ hybridization. In this way it is possible to determine the distribution of specific receptors in a tissue by accurately determining the distribution of mRNA that encodes for the receptor protein. This is a particularly valuable technique for the administration of psychotropic drugs. 

Use of Human Tissue Slices in Drug Targeting Research... 

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