Tissue, intact

Parameters used to measure toxicity of cultured cells can be general properties related to loss of vital functions or impairment of cell-type-specific functions. These indicators can be further classified as those occurring shortly after toxicant exposure and those requiring prolonged culture for expression. 

Function of mitochondria is also commonly monitored as an indicator of cellular toxicity. Mitochondrial uptake and retention of the fluorescent dye, rhodamine 123, can be visualized microscopically. Biochemical measurements of mitochondrial function include the ATP-ADP ratio and dehydrogenase activity with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), which yields a colored formazan product upon reduction. The dye, neutral red (3-amino-7-dimethyl-amino-2-methylphenazine hydrochloride), targets lysosomes, and its retention is inversely related to cytotoxicity. Commercially available versions of the MTT and neutral red assays have been adapted to microtiter plate formats to provide highly efficient screening assays. Examples of how cell-type-specific functions can be followed as indicators of cell toxicity are included in Table 8.1. 

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C12H20O4. M.p. 165°C. A plant growth hormone, which is produced in damaged plant tissue, and on diffusing into adjacent intact tissue cells stimulates them to divide. Traumatic acid has been isolated from the pods of green beans. 

Table 3). For example, arabinose and xylose differ from ribose only in the orientation of the 2 - and 3 -OH groups yet exhibit markedly different potencies. Whereas 9-(tetrahydrofuryl)-Ade ( SQ 22,536) and 9-(cyclopentyl)-Ade are without hydroxyl groups and are less potent, they offer metabolic and biochemical stability useful for many types of studies. It is, however, the removal of two of the hydroxyl groups, that elicits the largest improvement in inhibitory potency, in particular the 2, 5 -dideoxy- modification (Table 3). With these improvements in potency, these cell permeable compounds, in particular 2, 5 -dd-Ado, have become useful research tools and have been used to inhibit adenylyl cyclases and to lower cAMP levels and alter function in numerous studies in isolated cells or intact tissues. 

Animal cell cultures that are initiated from cells removed directly from the animal are called primary cultures (Figure 2). Primary cultures include both explant cultures (i.e., cultures initiated from small pieces of intact tissue), as well as cultures initiated from preparations of individual or dispersed cells (obtained from intact tissue by mechanical or proteolytic dismption). Nerve fiber explant cultures in blood plasma were among the earliest types of tissue cultures (Harrison, 1907). Cells grow out from such tissue explants and form a single layer of cells completely filling the tissue culture vessel surface. Such cell cultures are called confluent monolayers. Confluent monolayers can then be treated with trypsin, so as to remove the individual cells from the culture vessel surface. The resulting cell suspension is then transferred into other culture containers, so that more viable monolayer... 

Because of their strategic localization, astrocytes play a crucial role in maintaining the extracellular ionic homeostasis, provide energetic metabolites to neurons and remove excess of neurotransmitter in schedule with synaptic activity. In addition, the strategic location of astrocytes allows them to carefully monitor and control the level of synaptic activity. Indeed, number of papers during the last 15 years have shown that cultured astrocytes can respond to a variety of neurotransmitters with a variety of different patterns of intracellular calcium increases (Verkhratsky et al. 1998). Later on, studies performed in intact tissue preparations (acute brain slices) further established that the plasma membrane receptors can sense external inputs (such as the spillover of neurotransmitters during intense synaptic activity) and transduce them as intracellular calcium elevations, mostly via release of calcium from internal stores (Dani et al. 1992 Murphy et al. 1993 Porter and McCarthy... 

Moran, R. and Porath, D., Chlorophyll determination in intact tissues using N,N-dimethytformamide. Plant Physiol., 65, 478, 1980. 

Saito, K. and Kawasaki, H. Comparative studies on the distribution of quinoidal chalcone pigments in extracts from insect wastes and intact tissues of dyer s saffron florets, Zeits. Lebens, Unter. Forsch., 194, 131, 1992. 

With these reservations in mind, we will next consider three approaches that have been used in the past to measure the efficacy of a partial agonist acting on an intact tissue. Each will be analyzed in two ways with the details given in Appendix 1.4C (Section 1.4.9.3). The first is of historical interest only and is based on Stephenson s original formulation, as expressed in Eq. (1.27) (Section 1.4.2) and with receptor occupancy given by the Hill-Langmuir equation in its simplest form, which we have already seen to be inadequate for agonists. The second analysis defines receptor occupancy as all the receptors that are occupied, active plus inactive. 

A. Analysis of Method 1 (Section 1.4.8) proposed for the determination of the efficacy of a partial agonist acting on an intact tissue ... 

Some antagonists combine the ability to block open ion channels with a competitive action at or near the agonist binding site. A well-characterized example is the nicotinic blocker tubocurarine (see Chapter 6). Agonists may also be open channel blockers, thus limiting the maximal response that they can elicit. Such agents (e g., decamethonium) may therefore behave as partial agonists when tested on an intact tissue ... 

So far, we have reviewed the various ways in which complex dose-response curves in intact-tissue bioassays can be the result, the pharmacological resultant, of two or more interacting activities. Now, if all that these bioassays achieved was to blur and obscure the underlying activities, they would have to give way to the newer, analytically simpler assays based on chemistry and biochemistry. However, the beauty of intact-tissue bioassays is that they are analytically tractable by using families of dose-response curves and appropriate mathematical models, the complexity of intact hormone-receptor systems can, indeed, be interpreted. Bioassay allows them to be studied as systems in ways denied to simple biochemical assays. 

Neither 79 nor 80 are detected in the intact tissue of the sponge implying that substrates and enzymes for the conversion are stored in different cellular compartments or different cell types. When isofistularin-3 (76) is the substrate, 77 is detected as a second cleavage product of this reaction. This wound-activated bioconversion is paralleled by a marked increase of biological activity of the products 79 and 80 compared to the isoxazolines. In feeding assays the aqueous... 

Protoplasts versus intact tissues Which cell-type to FRET... 

Although numerous mechanisms have been put forth to explain antigen unmasking by AR methods1,7,8 they remain speculative because intact tissue... 

Cultured and isolated cells 8.6.13 Intact tissues and organs... 

Lifetime of nitric oxide is an important parameter of its reactivity. Measurement of NO in intact tissue yielded a value in the order of 0.1 s [35] although preliminary estimates gave a much bigger lifetime. It has been accepted that the main reason for the rapid disappearance of NO in tissue is its reaction with dioxygen, which proceeds in aqueous solution with the following overall stoichiometry ... 

The 5-HTlc receptor appellation is vacant because, as described above, this receptor was renamed the 5-HT2C receptor due to structural, operational and transductional similarities with the 5-HT2 receptor family. A lower case appellation is used for the 5-ht1E and 5-ht1F receptors because a physiological role for these receptors in intact tissue has not been found [28]. 

The 5-HT4, 5-hts and 5-HT7 receptors are coupled to the stimulation of adenylyl cyclase. 5-HT4, 5-htg and 5-HT7 receptors preferentially couple to the stimulation of adenylyl cyclase, increasing cAMP formation, via the Gs family of G proteins (see Chs 19 and 21). These receptors, however, share only >35% overall sequence homology. For this reason, they are classified as distinct receptor groups or classes and not subtypes of a family. The grouping of these receptors together is considered to be somewhat arbitrary and may be modified in the future. A lower-case appellation is used for the 5-ht6 receptor because a physiological role for these receptors in intact tissue has not been found [28]. 

Blaustein Clearly, we want to study things in intact tissue, preferably even in situ. But there are some things that we simply cannot do at the level that we would like to look in intact tissue. That is why we use model systems. 

Wray Can we take the discussion one step back We have heard about the horrors of cultured cells, and the difficulty of doing anything in intact tissue. Ted Burdyga, you have comparisons of single cells and intact bundles. How robust a model are single cells for the intact muscle preparations ... 

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