Cell preparation fixation processes

It obviously was difficult to establish details of the N2 fixation process with intact organisms, as the ammonia fixed was rapidly assimilated into other compounds. So a search was initiated in several laboratories for a cell-free preparation that would fix N2. 

A number of techniques are available, including laser microprobe mass analysis (LAMMA) and various microprobes, that are capable of determining within tissues or cell preparations the cellular and subcellular distribution of lead in situ. While these techniques generally require varying levels of sample preparation (e.g., tissue fixation, cell suspensions, etc.), they provide powerful tools for probing different cellular compartments and processes involving lead. Thus, the site-specific distribution of lead can be used to evaluate local toxicity, which can be correlated with pathological alterations in tissues. 

Finally, the localizations of low-molecular-weight compounds requires special specimen preparation techniques, as these compounds are often diffusible, water- or organic-solvent soluble, and solubilized by conventional fixation and dehydration procedures. The reader is referred to ref. (12) for the processing of cells and tissues for the cytochemical and histochemical localization of these compounds. 

In their 1960 paper, Carnahan et al. reported that ATP was inhibitory to nitrogenase activity in their cell-free preparations. Hence, when McNary and Burris [24] reported that ATP was needed to support fixation, the report was met with a good deal of skepticism. But experiments in a number of other laboratories verified the absolute need for ATP. Not only is ATP needed, it is needed in substantial amounts. Under ideal conditions 16 ATP are required per N2 reduced to 2 NH3. Under normal conditions in nature the requirement probably is in the 20 to 30 ATP per N2 range. N2 reduction is energy demanding whether it is accomplished chemically in the Haber process or enzymatically by the nitrogenase system. 

Raman spectroscopy can offer a number of advantages over traditional cell or tissue analysis techniques used in the field of TE (Table 18.1). Commonly used analytical techniques in TE include the determination of a specific enzyme activity (e.g. lactate dehydrogenase, alkaline phosphatase), the expression of genes (e.g. real-time reverse transcriptase polymerase chain reaction) or proteins (e.g. immunohistochemistry, immunocytochemistry, flow cytometry) relevant to cell behaviour and tissue formation. These techniques require invasive processing steps (enzyme treatment, chemical fixation and/or the use of colorimetric or fluorescent labels) which consequently render these techniques unsuitable for studying live cell culture systems in vitro. Raman spectroscopy can, however, be performed directly on cells/tissue constructs without labels, contrast agents or other sample preparation techniques. 

Further proof that COj is assimilated by means of the enzyme oxalacetate /3-carboxylase was obtained by Evans and coworkers, who succeeded in preparing a cell-free preparation of this enzyme from liver. The enz3rme was able to catalyze the decarboxylation of oxalacetate to pyruvate. These investigators were able to demonstrate an uptake of C Oj. Utter and Wood have demonstrated conclusively, however, that in the presence of isotopic CO2, pyruvate can be converted to oxalacetate containing isotopic carbon and that the process is, of course, reversible. Addition of adenosine triphosphate to this liver enzyme system increased the rate of incorporation of C Os. Wood, Vennesland and Evans S have also shown that during the fixation of C02, isotopic carbon is incorporated solely and in equal concentrations into the carboxyl groups of pyruvate, lactate, malate and fumarate. 

In the following period of fifty years litde progress was made towards elucidating the mechanism of the process. Many attempts were made to obtain cell-fi ee preparations containing the enzymes presumably involved in nitrogen fixation, but these were unsuccessful until 1960. The intracellular orgeinisation of enzyme systems is known to be complex, groups of enzymes involved in sequential reactions... 

The economic importance of nitrogen-fixation by leguminous plants has led to extensive study of the process of nodulation and in particular the nature of the host specificity of Rhizobium. Antiserum raised to clover tissue reacts with virulent strains of R. trifolii and to a lesser extent with avirulent strains of the same organism. If the antiserum is pretreated with avirulent cells a purified antibody, specific for the virulent strain, can be prepared. The purified antisera will bind to R. trifolii virulent strains and clover cells, but not to incompatible Rhizobium species. Immunofluorescence can be used to demonstrate that antisera raised against the capsular polysaccharide of R. trifolii binds to the surface of clover root cells, in particular to the tips of the root hairs. Chemical analysis of this capsule shows that it... 

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