Protein status, determining

Protein status can be assessed by several biomarkers, including plasma amino acid profiles, albumin, and transthyretin (prealbumin) [42]. No marker alone is sufficient in determining status (Box 7.1). 

The overproduction of a wide variety of proteins has now been achieved in . coli and other cloning hosts. Many of these proteins are in chnical trials and, as indicated earlier, over a dozen are already on the market. The current status of many of these proteins is summarized in Table 24.2. The efficacy of mai r of the proteins listed remains to be determined because until the advent of recombinant DNA technology sufficient quantities were not available to enable clinical trials to be undertaken. It should be noted that clinical efficacy alone is not sufficient. Maiket size isjust as important since it can cost up to 50 million to bring a new dmg to the market place and company shareholders expect a good return on their investment. 

The major determinant of myocardial redox status is the glutathione content of the heart (Griffith and Meister, 1979). Therefore, fluctuations in myocardial glutathione status may exert a regulatory role in cellular metabolism in a comparable manner to the phosphorylation and dephosphorylation of proteins and enzymes. 

If cellular redox state, determined by the glutathione status of the heart, plays a role in the modulation of ion transporter activity in cardiac tissue, it is important to identify possible mechanisms by which these effects are mediated. Protein S-,thiolation is a process that was originally used to describe the formation of adducts of proteins with low molecular thiols such as glutathione (Miller etal., 1990). In view of the significant alterations of cardiac glutathione status (GSH and GSSG) and ion-transporter activity during oxidant stress, the process of S-thiolation may be responsible for modifications of protein structure and function. 

Since the MHLW designated shrimp/prawn and crab for mandatory labeling in June 2008 due to the almost unlimited use of crustacean in the processed foods in Japan and the status as a frequent cause of adverse food reactions in allergic patients, two ELISA methods for the determination of crustacean protein in processed foods have been developed (Seiki et al, 2007 Shibahara et al., 2007) EA test EIA-Crustacean [Nissui] produced by Nissui Pharmaceutical Co., Ltd. and Crustacean Kit [Maruha ] produced by Maruha Nichiro Eoods, Inc. Both kits have been validated according to the Japanese validation protocol (Sakai et al., 2008) and are commercially available. All the commercial ELISA kits are shown in Table 4.9. 

The protein content of milk is primarily influenced by the breed of cow, the stage of lactation, type of diet being fed and the health status of the cow, and is important in processing because the protein (and specifically casein) content of milk determines its cheese yield. Milk provides a highly digestible source of protein for a large proportion of the world s population, either as raw milk or processed into dairy products. In addition to this basic nutrition, milk... 

In summary, the de novo isolation of the cDNAs encoding enzymes of alkaloid biosynthesis is still achieved by using a variety of classical techniques, such as protein purification followed by partial amino acid sequence determination, and by newer techniques such as proteomics coupled to functional heterologous expression. The current status of cloned cDNAs specifically related to isoquinoline alkaloid biosynthesis is schematically presented in Figure 10.8. New additions to this list will certainly be made in the future as a result of a combination of approaches both new and old. 

During the monitoring of any protein in cerebrospinal fluid, it is necessary to bear in mind the functional status of the blood-CSF barrier, serum concentration of the respective protein, and the dimensions of the molecule (or the information based on the molecular weight). These data principally influence the resulting concentration in cerebrospinal fluid. It must also be emphasized again that the parallel determination of albumin in cerebrospinal fluid and serum is necessary. 

A number of methods are available for analyzing tumor HER-2 status. The selection of the method depends on the target molecule to be detected. The target molecules are DNA mRNA, and protein (Fig. 12.2). HER-2 gene amplification can be detected by Southern blot (Press et al., 1994), slot blot (Naber et al., 1990), and dot blot assays (Descotes et al 1993), fluorescence in situ hybridization (FISH) (Persons et al., 1997), in situ hybridization (ISH) on isolated nuclei or tissue sections (Smith et al., 1994), and polymerase chain reaction (Gramlich et al., 1994). Assays to determine mRNA oveiexpiession include Northern blot (Slamon et al., 1989), Western blot (Press et al., 1994), slot blot (Naber et al., 1990) and ISH (Naber et al., 1990). Methods to assess HER-2/mcm protein product overexpression... 

Enzyme-linked immunosorption assay can be used to measure HER-2 protein in tissue homogenates or in serum. It is a relatively simple technique and is well suited to automation (van de Vijver, 2001). However, when tumor cytosolic fractions are used, histological information is lost and invites the undesirable dilution effect. Therefore, the ELISA assay is not used routinely to determine HER-2 status. 

In addition to Ras, a number of other proteins are known to be substrates for FTase (many of unknown identity or function, determined by 2-D gel shift experiments in the presence and absence of FTI) these may also play a part in determining relative susceptibility to FTase inhibition.46 In particular, RhoB, a farnesylated protein involved with the formation of actin stress fibers and cytoskeletal organization, has been implicated in the growth-inhibitory consequences of FTase inhibition.47,48 Another study using the FTI 19 found that growth inhibition in soft agar of a panel of human tumor cell lines correlated with the mutational status of the H-and N-Ras proteins, but not with K-Ras mutations.49 In this study also, cells with genetic defects in addition to ras were sensitive to FTase inhibition. 

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