Amino acid analysis data evaluation

In this chapter, we provide protocols to determine the ability of a peptide to mediate DNA internalization in cultured human tumor cells. Fluorescence-assisted cell sorting (FACS) analysis is used to obtain quantitative data on the time and temperature dependence of macromolecular delivery. Confocal microscopy is used to study the subcellular localization in both fixed and live cells. Fluorescently labeled transferrin and dextran are used to label the clathrin-dependent (15) and the non-clathrin, non-caveolar (16) endocytic compartments, respectively. Expression of a caveolin-l-YFP fusion protein is used to label cell surface caveolae and intracellular caveosomes (17). Finally a protocol, for the overexpression of dominant-negative dynamin [GTPase deficient dynamin-2 containing the amino acid substitution K44A (18)] is provided to evaluate the dynamin dependence of the uptake mechanism. 

Glucose was the only major sugar and IMP and GMP were the only major nucleotides found. A sensory evaluation of the different processed products Indicated a preference for the drum dried product over the freeze or spray dried product. This preference could not be explained from sugar or nucleotide values and the amino acid data was Inconclusive. Since the authors have amassed such a large data pool for both volatile and nonvolatile compounds 1t Is unfortunate that some form of data analysis such as multlvarlent statistical analysis was not applied so as to determine which compounds were primarily responsible for the perceived flavor preference. 

ABRF-96SEQ offered a unique opportunity for participating laboratories ability to interpret the same data. There were 95 participants in the study, of which 98% perform sequence analysis, 54% use mass spectrometty routinely, and 64% provide an internal sequence analysis service. The total number of sequence reviewers were 157 or 1.65 per response and the total years of experience were 1061.4 or 6.8 years per reviewer. Concerning everyday sequence calling, 37% report only one person reviews the data while only 17% use more than 1 person 72% say that a simple sequence is called by one person and that complex data is evaluated by more than one reviewer. There were 165 protein sequencers in the responding laboratories and are distributed as follows 133 Applied Biosystems/Perkin Elmer, 22 Hewlett-Packard, 8 Porton/Beckman, and 2 MiUigen. Respondents reported the most difficult amino acids to identify were Cys (64.5%), Trp (55%), and Ser (22.5%). 

A crucial point in evaluating uptake data for amino acids is whether this uptake is mediated by a low-affinity system alone, or by a low- and a high-affinity system, i.e., if the uptake is mediated by one or two transport carriers. Shank and Campbell (1984) calculated the K , and Vmax values with the Pennzyme computer program (Kohn et al., 1979). This program uses a weighted nonlinear regression analysis, and Eadie-Hofstee plots are used to evaluate the presence of a two-carrier transport system. Figure 1... 

Expression of the inserted DNA is taken into account in order to evaluate the risk/safety of the new gene products on food, feed and the environment. Expression of vector backbone sequences and new ORFs may also be considered. Data obtained through molecular analysis should reveal whether the inserted vector DNA can be transcribed and translated. If potential new ORFs are identified, bioinformatics tools can assist to determine the likelihood of RNA formation, the possibility for transcription and translation to occur and the amino acid sequence of the putative new protein. If it is found that new proteins are likely produced, their potential impact on risk/safety should be fully characterised. The risk/safety assessment of any new protein is outside the scope of this document. 

---