Step 2 Cultural analysis

The second step is therefore to analyze the culture and climate in the organization in order to detect any characteristics that may impede the successful implementation of the requirements. It is often difficult for those inside the organization to be objective in per- 

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Metabolic control analysis (MCA) assigns a flux control coefficient (FCC) to each step in the pathway and considers the sum of the coefficients. Competing pathway components may have negative FCCs. To measure FCCs, a variety of experimental techniques including radio isotopomers and pulse chase experiments are necessary in a tissue culture system. Perturbation of the system, for example, with over-expression of various genes can be applied iteratively to understand and optimize product accumulation. 

Analysis of RMs enables assessment of laboratory factors (equipment, staff etc.) on quality, but it is the awareness of the importance of reference materials within the quality culture of the laboratory (recognizing crucial steps in method vahdation, ambitions to have good IQC and ambitions to have better performance scores in EQA) - that they are used to greatest effect. 

An interesting variation on the whole-cell MALDI approach was recently reported in a study aimed more at FTMS than TOF MS, but the results are nevertheless interesting and important to users of both methods for analysis of bacteria 40. Wilkins s group showed both MALDI-TOF and MALDI-FTMS spectra of whole bacteria grown on isotopic media depleted in C13 and N14. Because most bacterial identification protocols involve a culture step prior to analysis, it is possible to manipulate the sample based on control of the growth media. For mass spectral analysis manipulation of the isotope profile... 

The most common criticism of whole-cell MALDI is that the method requires a relatively large number of cells, usually obtained directly from culture media. In principle, an analysis of even a few unknown bacteria (a colony-forming unit) is possible after a culture step. More important is the number of bacteria needed in a sample or on the sample probe for successful analysis. Detection of a very small number of bacteria could eliminate the need for a preliminary culture step. This would be a considerable asset for environmental analysis (unless to many bacteria were detected) and for the detection of a bioterrorism-related release of bacteria. 

Electrospray (ESI) ionization mass spectrometry also plays in important role in bacterial characterization. Because it typically includes a chromatographic separation step, the approach is not considered as rapid as MALDI approaches, which do not incorporate a separation. However, compared to the times needed to grow bacteria in culture prior to analysis, the time frame is not lengthy, and the addition of chromatographic separation provides many opportunities to increase specificity. ESI/MS has been used to characterize cellular biomarkers for metabolic, genomic, and proteomics fingerprinting of bacteria, and these approaches are reported in two chapters. 

The sulfur-specific pathway for desulfurization of benzothiophene (BT) has been reported in Gordonia sp. Strain 213E. The metabolites of BT conversion were determined by ethylacetate extraction of the culture medium followed by GC-MS analysis [33,34], The reaction mechanism was proposed to be very similar to that of DBT for the first two steps (Fig. 4) however, the third step was quite different. 

The following basic protocols may be used alone or combined with other staining procedures in multiparameter flow cytometry experiments. Although they are illustrated with data from cells that proliferate in suspension, these protocols may be easily modified for the analysis of cells isolated from tissues or adherent cells in culture, by incorporating an initial step for the preparation of single cell suspensions. The assays are conducted at room temperature, unless otherwise noted. 

The premise establishing that each cultural good is unique and irreplaceable, and as consequence must be preserved as intact as possible, restricts the conditions for applying analytical procedures. Thus, the four steps comprised in the complete process of analysis, as shown in Scheme 1.3, are conditioned by this singular character inherent to monuments, art, and archaeological objects. 

Nevertheless, there are many questions still open because of problems to detect enzyme activities corresponding to each step of the pathway. The model of biosynthesis pathway was put together by studying the metabolism of exogenously applied intermediates in cell cultures of various origins and combining these results with data of native brassinosteroid patterns. It is more or less accepted that there are three pathways in parallel, the early and the late C6 oxidation pathway, as well as the 24/ -epimers follow ing the same route. Some observations in the analysis of native brassinosteroid patterns suggest a possible connection between the pathways. It was shown that seeds of Arabidopsis contain castasterone and 24-epi-brassinolide [34]. Also members of both 24-epimers, brassinolide and 24-epi-brassinolide were detected in tomato seeds [Winter, unpublished]. 

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. 

A lot of effort as regards phenomena identification is based on specific growth rate analysis. In Chapter 2, cell growth and division were proposed in three phases, based on values of specific growth rates. The number of steps observed in a culture depends on each system, that is, some steps may not exist or have an insignificant duration under some conditions. 

More recently, this model has undergone substantial modification. First, it became apparent from biosynthetic studies on cells in culture that there were two distinct B chains, B1 and B2 (Cooper et al., 1981), which were not well resolved in laminin prepared from the EHS tumor. Analysis of the stoichiometry of the chains produced in these systems suggested that the laminin molecule contained one A, one Bl, and one B2 chain (Fig. 9). Studies with a monoclonal antibody specific for the A chain indicated that the A chain formed part of the short arm as well as the long arm of laminin (Palm et al., 1985). The assembly of the molecule appears to proceed in discrete steps, with the initial assembly of a B1-B2 dimer linked by disulfide bonds to which an A chain is added (Morita et al., 1985 Peters et al., 1985). 

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