Cells, biological fractionation

The magnification of small things is a necessary facet of biological research. The most basic microscope is the light microscope which employs visible light to amplify and detect small objects (Fig. E.l). Other specialised microscopes which are used are fluorescence and confocal and electron microscopes. In cell biology and biochemistry, one uses a microscope especially to check cells from tissue culture, tissue sections, as well as subcel-lular fractions. 

Methods to separate or fractionate biological and biomedical samples lie at the heart of a diverse range of scientific disciplines including biochemistry, cell biology and molecular biology. The choice and mode of separation is important to obtain the best results and avoid artefacts. There are a number of physical and chemical separation technologies routinely used by the bioanalytical chemist that are described in this chapter and various other chapters of this textbook. 

The use of elicitors can significantly enhance the production of metabolites. The elicitors are divided mainly in two groups. The biotic elicitors which are compounds of biological origin (e.g. fungal spores, fungal cell wall fractions, cellulase, chitosane) and the abiotic elicitors which include metal ions, high salt concentrations, UV radiations, sonication. Treatment of Hyoscyamus muticus hairy roots with 50-500 jig/ml of chitosane resulted in a 5-fold increase in the accumulation of hyoscyamine [84]. Similar results were obtained by Halperin and Flores [85] who obtained, with hairy roots of the same species, hyoscyamine up to 6-fold when elicited with mannitol. 

Knowledge about the subcellular localization of a protein may provide a hint as to the function of the protein. The combination of classic biochemical fractionation techniques for the enrichment of particular subcellular structures with the large-scale identification of proteins by mass spectrometry and bioinformatics provides a powerful strategy that interfaces cell biology and proteomics, and thus is termed subcellular proteomics. ... 

Table 16.1. Biological mechanisms other than repopulation which may result in an increase of radiation resistance of tumour stem cells during fractionated radiotherapy and thereby contribute to the time factor...

As can be seen the distribution is almost the same the second time. This indicates that the fibers in this very sample are rather homogeneous and distribute statistically between the phases weighted according to their affinity for the phases. Had the sample consisted of various fractions a repeated distribution would not give the same results. For heterogeneous samples repeated distribution of course gives the possibility for fractionation. This is the basis for much of the use of this method in the field of cell biology. 

Figure 2. Distribution of acid hydrolases after fractionation of rat thoracic duct lymphocytes by zonal centrifugation. Histograms were plotted according to Bowers (4). Reproduced with permission from the Journal of Cell Biology.

In principle all studies to measure interaction between two molecules are affinity interactions. What is described imder this heading could also be described as traditional cell biological or biochemical approaches to measure binding between two systems. It was historically started with the observation of whole cells interacting, before more sophisticated methods of cell fractionation and artificial reconstruction have progressed to the point that almost any molecule can be isolated and packed into a de novo assembly to test for its role in the intact system. 

FIGURE 82.1 Schematic view of skin which highlights the epidermis, the basement membrane interleaved between the epidermis and the dermis, and the dermis underneath. Only a small fraction of the thickness of the dermis is shown. (Redrawn with permission from Darnell J.E., Lodish H.F., and Baltimore D. Molecular Cell Biology, 2nd Ed., Scientific American Books, New York, Chapter 23, Figure 23-2, p.905,1990.)... 

Albertsson (Paiiition of Cell Paiiicle.s and Macromolecules, 3d ed., Wiley, New York, 1986) has extensively used particle distribution to fractionate mixtures of biological products. In order to demonstrate the versatility of particle distribution, he has cited the example shown in Table 22-14. The feed mixture consisted of polystyrene particles, red blood cells, starch, and cellulose. Liquid-liquid particle distribution has also been studied by using mineral-matter particles (average diameter = 5.5 Im) extracted from a coal liquid as the solid in a xylene-water system [Prudich and Heniy, Am. Inst. Chem. Eng. J., 24(5), 788 (1978)]. By using surface-active agents in order to enhance the water wettability of the solid particles, recoveries of better than 95 percent of the particles to the water phase were obsei ved. All particles remained in the xylene when no surfactant was added. 

The numerous separations reported in the literature include surfactants, inorganic ions, enzymes, other proteins, other organics, biological cells, and various other particles and substances. The scale of the systems ranges from the simple Grits test for the presence of surfactants in water, which has been shown to operate by virtue of transient foam fractionation [Lemlich, J. Colloid Interface Sci., 37, 497 (1971)], to the natural adsubble processes that occur on a grand scale in the ocean [Wallace and Duce, Deep Sea Res., 25, 827 (1978)]. For further information see the reviews cited earlier. 

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