Guidelines for the Validation and Use of Immunoassays for Determination of Introduced Proteins in Biotechnology Enhanced Crops and Derived Food Ingredients

Recent advancements in agricultural biotechnology have created a need for analytical techniques to determine introduced proteins in crops enhanced through modern biotechnology techniques. These proteins are expressed in plant tissues and may be present in food ingredients. Immunoassays are ideally suited for protein detection and may be used as both quantitative and threshold methods. Microplate ELISA and lateral flow devices are two of the most commonly used immunoassay formats for agricultural biotechnology applications. This paper provides general background information and a discussion of criteria for the validation and application of immunochemical methods to the analysis of proteins introduced into plants and food ingredients using biotechnology methods. It is the result of a collaborative effort of members of the Analytical Environmental Immunochemical Consortium. This collaborative effort represents the combined expertise of several organizations to reach consensus on establishing guidelines for the validation and use of immunoassays. Further, the paper offers developers and users a consistent approach to adopting the technology as well as aid in producing accurate and meaningful results.     

New model for analysis of dynamic contrast-enhanced MRI data distinguishes metastatic from nonmetastatic transplanted rodent prostate tumors.

Dynamic contrast-enhanced MRI (DCEMRI) data were acquired from metastatic and nonmetastatic tumors in rodents to follow the uptake and washout of a low-molecular-weight contrast agent (Gd-DTPA) and a contrast agent with higher molecular weight (P792). The concentration vs. time curves calculated for the tumor rims and centers were analyzed using the two-compartment model (TCM) and a newly developed empirical mathematical model (EMM). The EMM provided improved fits to the experimental data compared to the TCM. Parameters derived from the empirical model showed that the contrast agent washout rate was significantly slower in metastatic tumors than in nonmetastatic tumors for both Gd-DTPA (P < 0.03) and P792 (P < 0.04). The effects of the tumor on blood flow in "normal" tissue immediately adjacent to the tumors were evident: Gd-DTPA uptake and washout rates were much lower in muscle near the tumor (P < 0.05) than normal muscle farther from the tumor. The results suggest that accurate fits of DCEMRI data provide kinetic parameters that distinguish between metastatic and relatively benign cancers. In addition, a comparison of the dynamics of Gd-DTPA and P792 provides information regarding the microenvironment of tumors.     

Patellofemoral joint: kinematic MR imaging to assess tracking abnormalities

The patellofemoral joint was imaged with magnetic resonance (MR) in the axial plane while the knee was positioned from 0 degrees to 32 degrees of flexion (nine positions). These multiple sequential images obtained within the early phases of flexion of the knee were viewed in a "cine-loop" format, producing a kinematic study that clearly demonstrated the relationship of the patella to the trochlear groove. Four healthy subjects and one patient with known bilateral subluxing patellae were studied. The preliminary results suggest that kinematic MR imaging of the patellofemoral joint is potentially useful for the evaluation of patellar tracking abnormalities.     

Characterization of high-energy phosphate compounds during reperfusion of the irreversibly injured myocardium using 31P MRS

Phosphorus-31 magnetic resonance spectroscopy (MRS) was used to monitor regional changes in high-energy phosphorus compounds and intracellular pH during 60 min of acute regional ischemia (acute occlusion of left anterior descending artery) and reperfusion in open-chest cats using a 1.2-cm two-turn coil sutured to the myocardium. During the 60-min ischemic phase, phosphocreatine (PCr) intensity was reduced to 47 +/- 4.9% (mean +/- SE) of control (p less than 0.01) by 15 min postocclusion while adenosine triphosphate (ATP) intensity decreased more slowly with the decrease (66 +/- 5.6%) achieving significance (p less than 0.05) only at 60 min postocclusion. Inorganic phosphate (Pi) increased to a maximum of 397 +/- 42% of control (p less than 0.01) while the pH decreased progressively from 7.36 +/- 0.02 to 6.02 +/- 0.14 (p less than 0.01). After release of occlusion PCr intensity recovered to 86 +/- 12% of the initial control value at 15 min postreperfusion but showed a subsequent downward trend to 79 +/- 8.8%. The ATP did not recover but tended to decline further during reperfusion. The Pi intensity decreased to 260 +/- 38% of control while the pH increased to 7.01 +/- 0.23 by 15 min postreperfusion. Thus, the reperfused irreversibly injured myocardium is characterized by persistent depletion of PCr and ATP and elevation of Pi. Phosphorus-31 MRS provides a nondestructive method for characterizing the reperfused irreversibly damaged myocardium.     

T-cell acute lymphoblastic leukaemia with late developing Philadelphia chromosome

A case of childhood T-cell acute lymphoblastic leukaemia (ALL) is presented in which the only chromosome abnormality at diagnosis was a deletion of part of the short arm of one chromosome 9 (9p-). Cytogenetic studies at relapse showed, in addition to 9p-, a partial deletion of the long arm of one chromosome 6 (6q-) and the Philadelphia chromosome (Ph1) produced as a result of the classical translocation t(9q+;22q-). All metaphases from haemopoietic colonies grown from a cryopreserved specimen of this patient's marrow at relapse were normal, in contrast to haemopoietic colonies cultured from patients with chronic myelogenous leukaemia (CML) which contained the Ph1. A hypothesis which incorporates T-cell ALL with late development of the Ph1 into the overall family of Ph1 positive diseases is suggested.     

A Golgi fragmentation pathway in neurodegeneration

The Golgi apparatus processes intracellular proteins, but undergoes disassembly and fragmentation during apoptosis in several neurodegenerative disorders such as amyotrophic lateral sclerosis and Alzheimer's disease. It is well known that other cytoplasmic organelles play important roles in cell death pathways. Thus, we hypothesized that Golgi fragmentation might participate in transduction of cell death signals. Here, we found that Golgi fragmentation and dispersal precede neuronal cell death triggered by excitotoxins, oxidative/nitrosative insults, or ER stress. Pharmacological intervention or overexpression of the C-terminal fragment of Grasp65, a Golgi-associated protein, inhibits fragmentation and decreases or delays neuronal cell death. Inhibition of mitochondrial or ER cell death pathways also decreases Golgi fragmentation, indicating crosstalk between organelles and suggesting that the Golgi may be a common downstream-effector of cell death. Taken together, these findings implicate the Golgi as a sensor of stress signals in cell death pathways.     

Inactivation of purified human alpha 2-antiplasmin and purified human C1 inhibitor by synthetic fibrinolytic agents

3-Hydroxypropyl flufenamide (Flu-HPA) is one of a series of flufenamic acid derivatives that enhances blood clot lysis in vitro. Studies of possible mechanisms of action of Flu-HPA were undertaken. The profibrinolytic activity of Flu-HPA in clot lysis assays was found to be dependent on plasminogen. The influence of Flu-HPA on the ability of purified alpha 2-antiplasmin to inhibit purified plasmin was studied. Plasmin activity was determined using 125I-fibrin plates or the spectrophotometric tripeptide substrate, Val-Leu-Lys-paranitroanilide. At Flu-HPA concentrations greater than 1 mM, the inhibitory activity of alpha 2-antiplasmin was abolished in a time-dependent and concentration- dependent manner. The influence of Flu-HPA on the ability of purified Cl inhibitor to inhibit purified plasma kallikrein and beta-Factor XIIa was also studied. Cl inhibitor activity was abolished by Flu-HPA at concentrations greater than 2 mM. Notably, Flu-HPA up to 60 mM did not affect the amidolytic activities of plasmin, kallikrein, or beta-Factor XIIa. Flu-HPA did not release enzyme activity from preformed complexes of either alpha 2-antiplasmin and plasmin of Cl inhibitor and kallikrein. A water-soluble derivative of flufenamic acid, N-flufenamyl- glutamic acid, also inactivated alpha 2-antiplasm and Cl inhibitor. This inactivation was shown to be reversible. These results indicate that synthetic fibrinolytic compounds such as flufenamic acid derivatives may promote fibrinolysis by directly inactivating alpha 2- antiplasmin and Cl inhibitor.