Approaches to assess IgE mediated allergy risks (sensitization and cross-reactivity) from new or modified dietary proteins.

The development and introduction of new dietary protein sources has the potential to improve food supply sustainability. Understanding the potential allergenicity of these new or modified proteins is crucial to ensure protection of public health. Exposure to new proteins may result in de novo sensitization, with or without clinical allergy, or clinical reactions through cross-reactivity. In this paper we review the potential of current methodologies (in silico, in vitro degradation, in vitro IgE binding, animal models and clinical studies) to address these outcomes for risk assessment purposes for new proteins, and especially to identify and characterise the risk of sensitization for IgE mediated allergy from oral exposure. Existing tools and tests are capable of assessing potential crossreactivity. However, there are few possibilities to assess the hazard due to de novo sensitization. The only methods available are in vivo models, but many limitations exist to use them for assessing risk. We conclude that there is a need to understand which criteria adequately define allergenicity for risk assessment purposes, and from these criteria develop a more suitable battery of tests to distinguish between proteins of high and low allergenicity, which can then be applied to assess new proteins with unknown risks.

Broiler performance and carcass characteristics when fed diets containing lysine maize (LY038 or LY038 x MON 810), control, or conventional reference maize.

Lysine maize (Zea mays), LY038, was developed through the application of modern biotechnology to accumulate free Lys in the germ portion of maize grain and provide an alternative to direct addition of supplemental Lys to poultry diets. Maize LY038 x MON 810 was produced by conventional breeding of LY038 with MON 810, which provides the corn plant protection against feeding damage from the European corn borer. A 42-d broiler feeding study (10 pens of 10 male Cobb x Cobb 500 broilers/treatment) was conducted to compare the feeding value of grain from LY038 or LY038 x MON 810 to that of a conventional control (similar genetic background to the test maize) and 5 conventional maize hybrids. The LY038 and LY038 x MON 810 maize-based diets and control and conventional reference maize-based diets supplemented with l-Lys HCl were formulated to a Lys level below that required for optimal bird performance, whereas all other essential amino acids were present at levels, relative to Lys, above those required for optimal bird performance [1.05% and 0.90% total Lys (as-fed) for d 0 to 21 and d 21 to 42, respectively]. Total Lys level in control and reference maize-based diets without supplemental l-Lys HCl was formulated to be 0.079% lower than supplemented diets. Weight gain, feed efficiency, and carcass yield and composition of broilers fed diets containing LY038 or LY038 x MON 810 were not different (P > 0.05) from that of broilers fed l-Lys HCl-supplemented diets and were superior (P < or = 0.05) to that of broilers fed conventional maize diets without supplemental l-Lys HCl. Both broiler performance and carcass data demonstrate that the bioefficacy of the incremental Lys in LY038 or LY038 x MON 810 grain was not different from that of Lys in conventional maize diets supplemented with l-Lys HCl. Thus, LY038 and LY038 x MON 810 can be considered as wholesome as and more nutritious than conventional maize due to its higher-than-average Lys content.

Determining whether transgenic and endogenous plant DNA and transgenic protein are detectable in muscle from swine fed Roundup Ready soybean meal.

Questions regarding the digestive fate of DNA and protein from transgenic feed have been raised in regard to human consumption and commercial trade of animal products (e.g., meat, milk, and eggs) from farm animals fed transgenic crops. Using highly sensitive, well-characterized analytical methods, pork loin samples were analyzed for the presence of fragments of transgenic and endogenous plant DNA and transgenic protein from animals fed meal prepared from conventional or glyphosate-tolerant Roundup Ready (RR) soybeans. Pigs were fed diets containing 24, 19, and 14% RR or conventional soybean meal during grower, early-finisher, and late-finisher phases of growth, respectively, and longissimus muscle samples were collected (12 per treatment) after slaughter. Total DNA was extracted from the samples and analyzed by PCR, followed by Southern blot hybridization for the presence of a 272-bp fragment of the cp4 epsps coding region (encoding the synthetic enzyme 5-enolpyruvylshikimate-3-phosphate synthase derived from Agrobacterium sp. strain CP4) and a 198-bp fragment of the endogenous soybean gene le1 (encoding soy lectin). Using 1 microgram of input DNA per reaction, none of the extracted samples was positive for cp4 epsps or le1 at the limit of detection (LOD) for these PCR/Southern blot assays. The LOD for these assays was shown to be approximately one diploid genome equivalent of RR soybean DNA, even in the presence of 10 micrograms of pork genomic DNA. A 185-bp fragment of the porcine preprolactin (prl) gene, used as a positive control, was amplified from all samples showing that the DNA preparations were amenable to PCR amplification. Using a competitive immunoassay with an LOD of approximately 94 ng of CP4 EPSPS protein/g of pork muscle, neither the CP4 EPSPS protein nor the immunoreactive peptide fragments were detected in loin muscle homogenates from pigs fed RR soybean meal. Taken together, these results show that neither small fragments of transgenic DNA nor immunoreactive fragments of transgenic protein are detectable in loin muscle samples from pigs fed a diet containing RR soybean meal.

Attempts to detect transgenic and endogenous plant DNA and transgenic protein in muscle from broilers fed YieldGard Corn Borer Corn.

Questions regarding the digestive fate of DNA and protein from transgenic grain have been raised in regard to human consumption and trade of animal products (e.g., meat, milk, and eggs) from farm animals fed transgenic crops. Using highly sensitive, fully characterized analytical methods, fragments of transgenic and endogenous plant DNA, as well as transgenic protein, were not detected in chicken breast muscle samples from animals fed YieldGard Corn Borer Corn event MON 810 (YG). Total DNA was extracted from breast muscle samples from chickens fed for 42 d with a diet including either 55 to 60% YG grain or 55 to 60% conventional corn grain. DNA preparations were analyzed by PCR followed by Southern blot hybridization for the presence of a 211-bp fragment of the Bacillus thuringiensis (Bt) cry1Ab gene and a 213-bp fragment of the endogenous corn gene sh2 (encoding ADP glucose pyrophosphorylase). By using 1 microg of input DNA per reaction, none of the extracted samples was positive for cry1Ab or sh2 at the limit of detection for these PCR assays. A 396-bp fragment of the chicken ovalbumin (ov) gene, used as a positive control, was amplified from all samples showing that the DNA preparations were amenable to PCR amplification. By using a competitive immunoassay with a limit of detection of approximately 60 ng of CrylAb protein per gram of chicken muscle, neither the CrylAb protein nor immunoreactive peptide fragments were detectable in the breast muscle homogenates from chickens fed YG grain.

Inhibition of cholesteryl ester transfer protein by apolipoproteins, lipopolysaccharides, and cholesteryl sulfate.

Cholesteryl ester transfer protein (CETP) mediates the exchange of cholesteryl esters and triglycerides between lipoproteins in the plasma. In studies dealing with the mechanism of CETP-mediated lipid transfer, we have examined the effects of several classes of biomolecules, including apolipoproteins and related synthetic peptides, cholesteryl sulfate, and lipopolysaccharides. In all cases, the molecules were inhibitory and their effects were associated with modifications of either HDL, LDL, or both. However, the probable mechanisms were distinct for each class of inhibitor. Inhibition of lipid transfer activity by apolipoprotein A-I was correlated with an increase in the apolipoprotein A-I content of HDL but not LDL, whereas the primary effect of cholesteryl sulfate was associated with modification of LDL, and only modest alteration of HDL. Lipopolysaccharides were found to modify the size and charge properties of both LDL and HDL over the same concentration ranges that affected CETP activity, but might also interact directly with CETP. It is suggested from the present studies that a variety of biomolecules that can interact with lipoproteins under natural or pathological situations have the potential to modify CETP activity, which in turn could affect normal lipoprotein composition and distribution.

Spi-1: an hepatic serine protease inhibitor regulated by GH and other hormones.

A sensitive RNAse protection method was used to show that serine protease inhibitor-1 (Spi-1) is expressed in rat liver and heart, but not in kidney or brain. Bovine somatotropin (bGH) and placental lactogen (bPL) induced rat hepatocyte cultures to express both Spi-1 and IGF-1 mRNA, with bPL approximately 100-fold more potent than bGH. Bovine prolactin (bPrL) did not induce hepatocyte Spi-1 mRNA, demonstrating lack of involvement of lactogenic receptors. Albumin mRNA levels were stable during hepatocyte culturing and were unaffected by growth hormone (GH) treatment, showing that neither culture conditions nor GH treatment affected cellular differentiation. Eliminating serum-free medium hormone supplements one at a time, estradiol, testosterone and T3 were shown to be unnecessary for GH induction of Spi-1, while dexamethasone removal decreased Spi-1 mRNA levels to 10% of GH-stimulated controls. bGH induction of Spi-1 mRNA in the presence of only dexamethasone and glucagon was 75% higher (p < 0.01) than levels seen with insulin also present.

Tissue repair by thrombin-derived peptides in the rat.

An ordered sequence of events occurs following tissue damage to bring about healing. One molecule involved in this process is thrombin. Utilizing rat linear incision and full dermal excision models, we have investigated the ability of two thrombin-derived RGD-containing peptides, p517 and p508, to enhance tissue repair under normal and healing-impaired conditions. p508, at 0.5 micrograms peptide per wound, produced a significant .23% improvement in wound strength in a dose-dependent manner. Similarly, a single application of 0.5 micrograms p517 per 6-cm linear incision would significantly increased wound-breaking strength approximately 18% at nine days postsurgery (control: 3.95 +/- 0.13 Newtons, vs. p517: 4.68 +/- 0.13 Newtons; P < 0.001). However, in glucocorticoid-stressed rats, the application of 0.5 micrograms per wound p508 or 517 did not significantly influence steroid-impaired healing. In the full dermal skin excision wound model, a single application of 0.5 micrograms p508 per wound at the time of surgery significantly reduced average wound area at days 3 and 5, when healing was impaired by glucocorticoid administration. Wound area was also reduced by p508 treatment at day 3 in the normal animal, but this effect was not statistically significant. We suggest wound-healing benefits of p508 and p517 may activate wound fibroblast proliferation or stimulate other cell types in the wound site through an RGD-mediated interaction.

Characterization of 3T3-L1 storage lipid metabolism: effect of somatotropin and insulin on specific pathways.

TLC of total cellular lipids showed that 3T3-L1 cells predominantly accumulate triglycerides. GH and insulin principally regulated glucose utilization for synthesis of the fatty acid portion of 3T3-L1 triglycerides, with little effect on glucose utilization for synthesis of other lipids or triglyceride glycerol. Gas chromatographic mass spectrometry (MS) showed that 50% of 3T3-L1 triglyceride fatty acids are 16-carbon chains. However, 3T3-L1 adipocytes were unusual, in that 20% of their triglyceride fatty acids were C15 or C17 in length, observed by both MS of saponified triglycerides and electrospray MS of intact triglycerides. Gas chromatographic analysis of 14C-labeled fatty acids showed 3T3-L1 utilization of [14C]glucose for synthesis of C15 and C16 fatty acids. Insulin and GH regulated the amount of [14C]glucose incorporation into C15 and C16 fatty acids without altering their relative ratio. GH antagonized all studied insulin-regulated events. GH antagonism of insulin-stimulated 3T3-L1 glucose transport, glucose oxidation, and glucose utilization for lipid synthesis reached a plateau of 50-60% inhibition at 0.1-0.2 nM. Insulin, at 0.1 nM, suppressed 3T3-L1 generation of glycerol from lipolysis by approximately 50%. GH, between 0.04-1.0 nM, fully reversed the insulin-inhibited lipolysis, although GH did not stimulate lipolysis beyond that in untreated control cultures. The results suggest that GH regulates a very early event in the insulin signal transduction pathway, such that is affects all insulin-responsive processes to essentially the same extent.

Acceleration of soft tissue repair by a thrombin-derived oligopeptide.

Augmentation of thrombin-modulated chemotaxis and mitogenic activity within the early phase of soft tissue repair is now possible. Identification of high-affinity thrombin receptor binding domains within thrombin has enabled the synthesis of a family of peptides which interact with thrombin receptors and enhance in vitro mitogenesis. A single (5.0 micrograms/wound) application of the thrombin receptor-activating peptide (P517-30) significantly increased wound breaking strength from Day 5 (31% over controls) to Day 12. Two models of impaired healing created by radiotherapy (RT) were used to elucidate possible mechanisms of P517-30 action. Although P517-30 did not completely overcome the RT-induced healing impairments, it increased breaking strength under conditions of penetrating whole body RT-induced pancytopenia by 22% and of nonpenetrating surface RT-induced dermal cell damage by 42%. This suggests that P517-30 directly stimulates resident endothelial cells, fibroblasts, or other cells to overcome dermal and circulating monocytic deficits. These results suggest a method to accelerate wound healing with potential clinical applications and emphasize the activity of thrombin as a growth factor.

Cellular mechanism of the insulin-like effect of growth hormone in adipocytes. Rapid translocation of the HepG2-type and adipocyte/muscle glucose transporters.

The cellular mechanism whereby growth hormone (GH) acutely stimulates adipocyte glucose uptake was studied in cultures of primary rat adipocytes differentiated in vitro. Preadipocytes were isolated by collagenase digestion of inguinal fat-pads from young rats and were differentiated in the presence of 3-isobutyl-1-methylxanthine, insulin and dexamethasone. The development of an adipocyte morphology (i.e. lipid inclusions) was observed over 6 days after initiation of differentiation. Coincident with this phenotypic change was an increase in glyceraldehyde-3-phosphate dehydrogenase (GPDH) activity and in cellular content of the HepG2-type (Glut1) and adipocyte/muscle (Glut4) glucose transporter isoforms as determined by Western immunoblotting of total cellular protein. Age-matched undifferentiated cells expressed the Glut1 transporter and low levels of GPDH, but neither accumulated lipid nor exhibited measurable expression of the Glut4 protein. On day 6 after the initiation of differentiation, GH and insulin stimulated 2-deoxy[14C]glucose uptake in a dose- and time-dependent fashion in adipocytes cultured under serum-free conditions for at least 15 h. Western-blot analysis of subcellular fractions revealed that both GH and insulin rapidly (within 20 min) stimulated translocation of the Glut1 and Glut4 proteins from a low-density microsomal fraction to the plasma membrane. Confirmatory evidence was provided in immunocytochemical experiments utilizing antisera directed against the C-terminal region of the Glut4 protein and a fluorescein isothiocyanate-labelled second antibody. Observation of the cells via confocal laser microscopic imaging was consistent with glucose transporter redistribution from an intracellular region to the plasma membrane after treatment with GH or insulin. On the basis of these data, we suggest that the insulin-like effect of GH on adipocyte glucose transport involves translocation of the Glut1 and Glut4 proteins to the plasma membrane. Furthermore, stimulation of glucose-transporter translocation by both GH and insulin may indicate a common cell signalling element between the adipocyte GH and insulin receptors or, alternatively, the existence of multiple cellular mechanisms for stimulating glucose-transporter translocation.

The carboxyl terminal segment of the c-Ki-ras 2 gene product mediates insulin-stimulated phosphorylation of calmodulin and stimulates insulin-independent autophosphorylation of the insulin receptor.

Cationic cofactors (e.g., polylysine or histone H2B) are necessary to observe phosphorylation of calmodulin in cell-free systems containing partially purified insulin receptors from a variety of tissues. The highly basic carboxyl terminus of the human c-Ki-ras 2 gene product stimulated both the in vitro phosphorylation of calmodulin and autophosphorylation of the beta-subunit of the insulin receptor, independently of insulin. Addition of insulin increased phosphate incorporation into calmodulin 2.5 fold. The K0.5 for insulin was approximately 5 x 10(-8) M. Maximal phosphorylation occurred at 120 microM c-Ki-ras 2 in the absence of Ca2+ and was inhibited by free Ca2+ concentrations above 0.1 microM. These data suggest the c-Ki-ras 2 gene product, an endogenous membrane protein, may play an important role in the cellular mechanism of insulin action.

Synthetic peptides bind to high-affinity thrombin receptors and modulate thrombin mitogenesis.

Initiation of cell proliferation by thrombin requires signals generated by thrombin interaction with specific high-affinity receptors and thrombin enzymic activity. Using synthetic peptides representing various domains of thrombin, we have identified a region adjacent to the proteolytic pocket of thrombin which confers high-affinity binding and generation of mitogenic signals. One peptide, representing residues 508 to 530 of human prothrombin (p508-530), inhibits up to 70% of the specific binding of 125I-alpha-thrombin at concentrations of less than 100 nM, enhances the ability of thrombin to stimulate DNA synthesis and stimulates DNA synthesis in cells treated with 25 ng/ml phorbol myristate acetate (PMA). Thus, this peptide or a portion of this peptide appears to represent the high-affinity receptor binding domain of thrombin. In contrast to the 23 amino acid peptide (p508-530), the tetrapeptide RGDA (p517-520) contained in this region competes for 125I-thrombin binding at concentrations from 100 to 2000 nM, but inhibits rather than stimulates the mitogenic effects of alpha-thrombin. Non-homologous peptides, or fibronectin-specific peptides (such as RGDS or GRGDSP) do not compete for 125I-alpha-thrombin binding and have no effect on thrombin mitogenesis. These studies demonstrate that peptides representing portions of the binding domain of thrombin: i) can generate receptor-occupancy related signals that enhance thrombin mitogenesis and are themselves mitogenic in cells treated with PMA; or ii) in the case of RGDA (which may be too small to generate signals), can act as antagonists, inhibiting the mitogenic effects of thrombin by preventing thrombin-receptor interaction.

Somatotropin antagonism of insulin-stimulated glucose utilization in 3T3-L1 adipocytes.

It is well established that somatotropin (GH) antagonizes insulin action in vivo and that supraphysiologic concentrations of GH frequently result in insulin resistance and glucose intolerance. However, the demonstration of an anti-insulin activity by GH in vitro has been difficult. This study, therefore, set out to determine whether cultures of 3T3-L1 adipocytes could be used to examine the anti-insulin activity of GH. The ability of insulin to stimulate glucose utilization by 3T3-L1 adipocytes increases approximately five-fold during the first 4 days following treatment of the cells with a differentiation medium. It was found that glucose utilization in 3T3-L1 adipocytes is regulated in a reciprocal fashion by insulin and GH. Bovine or human GH directly inhibit up to 50% of insulin-stimulated [14C]-glucose incorporation into lipids in a concentration-dependent manner. The 3T3-L1 sensitivity to GH appears to be at the maximum (50% inhibition of an insulin response) immediately following removal of the cells from the differentiation medium and remains essentially constant during the subsequent 4 days. The GH inhibition of insulin action does not appear to be due GH enhancement of cellular degradation of insulin, competitive binding of GH to the insulin receptor, or GH-induced decrease in cell number. The 3T3-L1 adipocyte system appears to be a sensitive and reliable in vitro model with which to study the molecular mechanisms involved in both GH antagonism of insulin action and development of hormone responsiveness during cellular differentiation into adipocytes.

Regulation of release of somatotropin from in vitro cultures of bovine and porcine pituitary cells.

Bovine and human GH releasing factors (GHRF), in concentrations ranging from 10 pM to 10 nM, stimulated GH release from cultured bovine and porcine anterior pituitary cells. Agents that increase intracellular cAMP levels (e.g. isobutylmethylxanthine and 8-bromo-cAMP) also stimulated bovine and porcine GH release. Somatostatin, in doses ranging from 1-100 nM, inhibited both basal and GHRF-stimulated GH release from the bovine pituitary cultures, and 100 nM somatostatin inhibited GHRF-stimulated release of porcine GH. Addition of exogenous bovine GH suppressed basal, but not GHRF-stimulated, release of bovine GH. Human insulin-like growth factor I did not suppress basal or GHRF-stimulated release of bovine GH from bovine pituitary cells, although it has been confirmed in this report that insulin-like growth factor I suppresses stimulated release of GH from rat cells. Furthermore, the GH release peptides described by Momany et al. stimulated little or no GH release from bovine or porcine pituitary cell cultures, in contrast to their activity in rat cells. The results show that whereas some regulatory features of GH release from bovine, porcine, and rat pituitary cell cultures are similar, others differ significantly.

Purification of recA-based fusion proteins by immunoadsorbent chromatography. Characterization of a major antigenic determinant of Escherichia coli recA protein.

Monoclonal antibodies to Escherichia coli recA protein were prepared, characterized, and used as affinity reagents for the purification of recA and recA:somatostatin fusion proteins. The monoclonal antibodies recognize an antigenic determinant or determinants located between amino acids 260 and 330 of recA. Addition of a fragment of the recA gene coding for these amino acids to an unrelated gene (beta-galactosidase) allowed the resulting beta-galactosidase fusion protein to be recognized by the recA monoclonal antibodies.

Human monocyte-derived growth factor(s) for mesenchymal cells: activation of secretion by endotoxin and concanavalin A.

Cultured peritoneal macrophages have previously been shown to release a potent mitogen for mesenchymal cells. Peritoneal macrophages are derived from peripheral blood monocytes, one of the principal inflammatory cells associated with numerous tissue responses to injury. Cultured human monocytes can be activated by endotoxin or concanavalin A to secrete a potent growth factor(s) that is active on human smooth muscle cells, human fibroblasts and 3T3 cells. The optimal conditions for activation of monocyte release of this monocyte derived growth factor(s) MDGF) were to expose 5-day-old monocyte cultures (initially plated at 6.8 X 10(5) cells/ml medium) to 10 microgram/ml endotoxin or 6 microgram/ml concanavalin A for approximately 20 hr. Monocytes can secrete MDGF into serum-free medium supplemented with 0.15% bovine serum albumin, MDGF stimulates both DNA synthesis and increase in cell number and is trypsin-sensitive, heat labile and nondialyzable. The relationship of MDGF to other monocyte products and its potential importance in wound repair and atherogenesis are discussed.

Hypersensitivity of platelets to thrombin: formation of stable thrombin-receptor complexes and the role of shape change.

The rate of rat platelet shape change increases sigmoidally with respect to thrombin concentration under conditions where free Ca++ and ADP are limited to prevent platelet aggregation. In addition, the rate of shape change due to thrombin is considerably enhanced when the platelets are first treated with concanavalin A (Con A), an agent which itself produces shape change. In the presence of both agents the rate is considerably greater than the sum of the rates due to Con A and thrombin separately. This suggests that shape change itself may trigger increased platelet sensitivity to thrombin. One possible mechanism through which this might occur is that shape change promotes binding of thrombin to its surface receptors. If so, then Con A-induced shape change might facilitate binding of 125I-thrombin. Initial binding studies using previously described methods showed that nonspecific trapping of 125I-thrombin, which is bound specifically to platelets during shape change, prevents accurate measurements of thrombin binding. We found, however, that a portion of 125I-thrombin which is bound specifically to platelets forms a stable complex with a 40,000 dalton platelet protein. This complex cannot be disrupted by boiling in SDS buffer containing 2-mercaptoethanol. Linkage of 125I-thrombin to this protein is specific for thrombin, since it can be competed for by an excess of unlabeled thrombin and because a similar complex does not form using 125I-trypsin. Concentrations of Con A that induce platelet shape change also markedly increase the amount of complex produced by a given thrombin concentration. In addition, colchicine, an inhibitor of Con A-induced platelet function, markedly inhibits formation of the 125I-thrombin-receptor complex. We suggest that the sigmoidal response to thrombin might be related to appearance of new thrombin receptors on the platelet surface.