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Native Glycoprotein II (Phaseolin, G-II), the major 7S storage protein from <i>Phaseolus vulgaris</i> seeds, var. 'Processor' is known to be resistant to <i>in vitro</i> proteolysis by most endopeptidases. On sequential treatments with pepsin and a mixture of trypsin and chymotrypsin, the sub-unit polypeptides of G-II were split midchain. The fragments produced however, retained reactivity with the antibody raised against native G-II quantitatively. When measured by rocket immunoelectrophoresis, the extent of <i>in vitro</i> degradation of G-II by these endopeptidases was negligible. This procedure was used for monitoring the <i>in vivo</i> or <i>in vitro</i> degradation of G-II by gut enzymes other than trypsin or chymotrypsin. Diets containing 10% of a highly purified G-II preparation, did not support growth of rats adequately. Faecal N outputs were elevated and the true N digestibility based on Kjeldhal estimation was only 37%. In contrast, the true GII-N digestibility, based on immunological estimations, was high. It is suggested that G-II and/or its limited breakdown fragments (by trypsin or chymotrypsin) are stimulants of endogenous N secretion in the small intestine. The higher extent of the degradation of G-II in the small intestine of rats <i>in vivo</i> than that obtained by pure endopeptidases <i>in vitro</i> suggested the presence in this tissue of other enzymes capable to act upon and modify the structure of G-II, prior to the action of trypsin and chymotrypsin. These other modifying proteolytic enzymes render the G-II molecule more negatively charged and more susceptible to the subsequent action of trypsin and chymotrypsin. It is suggested that protease content and the ratio of the concentration of the GII-modifying protease(s) to that of trypsin and chymotrypsin may vary appreciably along the small intestine. Accordingly, the dependence of the degradation of G-II <i>in vivo</i> on the competition between all the enzymes capable of attacking it during its passage through the gut may explain the variability of GII breakdown <i>in vivo</i>.

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Please read the abstract in the section 00front of this document / Dissertation (MSc Agric (Food Science and Technology))--University of Pretoria, 2006. / Food Science / unrestricted

BACKGROUND:The opaque2 mutant is valuable for producing maize varieties with enhanced nutritional value. However, the exact mechanisms by which it improves protein quality and creates a soft endosperm texture are unclear. Given the importance of improving nutritional quality in grain crops, a better understanding of the physiological basis for these traits is necessary.RESULTS:In this study, we combined transcript profiling and proteomic analysis to better understand which genes and proteins are altered by opaque2 in the W64A inbred line. These analyses showed that the accumulation of some lysine-rich proteins, such as sorbitol dehydrogenase and glyceraldehyde3-phosphate dehydrogenase, was increased in mature kernels and may contribute substantially to the lysine content of opaque2 endosperm. Some defense proteins such as beta-glucosidase aggregating factor were strongly down regulated and may be regulated directly by opaque2. The mutant also had altered expression of a number of starch biosynthesis genes and this was associated with a more highly crystalline starch.CONCLUSIONS:The results of these studies revealed specific target genes that can be investigated to further improve nutritional quality and agronomic performance of high lysine maize lines, particularly those based on the presence of the opaque2 mutation. Alteration of amylopectin branching patterns in opaque2 starch could contribute to generation of the soft, starchy endosperm.

Doctor of Philosophy / Department of Food, Nutrition, Dietetics, and Health / Brian L. Lindshield / Iron, vitamin A, and protein inadequacies are common in food-aid receiving countries, and maximizing nutrient intake and bioavailability are essential treatments. Fortified-blended foods (FBFs), are food-aid micronutrient-fortified legume-grain porridges distributed worldwide. FBFs have not consistently, effectively treated undernutrition, and it has been suggested that formulation and processing changes could improve their nutritional quality. Sorghum is a well suited FBF commodity, but high concentrations of ‘antinutritional’ tannin and phytic acid have limited its adoption. Iron bioavailability adaptation may be possible after long-term antinutritional factor consumption, but adaptive mechanisms are not well understood. In rats, salivary proline-rich proteins (PRPs) have been found to chelate tannins to improve iron bioavailability, this could be true for people as well. Several research design methods were employed to summarize FBF quality outcomes and the effect of tannin and phytic acid consumption on iron bioavailability. Extruded sorghum and corn FBFs were developed; protein quality, iron, and vitamin A outcomes were compared with a non-extruded corn-soy blend (CSB+) in rats. A narrative literature review and meta-analysis were conducted to determine tannin’s antinutritional effects on iron bioavailability, and the potential for adaptation through salivary PRPs. Two clinical trials examined the effect of long-term tannin or phytic acid consumption on iron bioavailability, salivary protein production, and correlations between PRPs and iron bioavailability. There were no differences between iron (hepatic iron 207-300 µmol/g *100), vitamin A (hepatic retinol 423-585.5 ng/mg), or protein quality (caloric efficiency: 101.3-113.3 g/kcal*100) between extruded FBFs regardless of commodity in rats. Compared to extruded FBFs, CSB+ caloric efficiency (49.0 ± 2.2 g/kcal*100) and growth (96.3 ± 3.4g vs. 208.6-236.6) were significantly reduced. A literature review suggested that there were differences in acute meal and long-term iron bioavailability with tannin consumption; tannic acid inhibited iron availability, while food-tannins did not. Meta-analysis suggested that tannin-PRP binding could protect iron bioavailability, that long-term tannin consumption did not significantly affect hepatic iron or non-heme iron absorption respectively in rats (d = -0.64-1.84; -2.7-0.13), and that PRP expression in rats during tannin consumption was correlated with improved iron bioavailability. There were no reductions in iron bioavailability or status based on long-term tannin (ps > 0.126) or phytic acid (ps > 0.08) consumption clinically, but basic PRP and cystatin subtypes were significantly correlated with improved iron bioavailability during tannin (ps < 0.03) and phytic acid (ps < 0.02) consumption. In vitro, it phytic acid-PRP binding did not occur, but phytic acid did specifically bind with cystatin SN, a non-enzymatic salivary protein. In conclusion, FBF formulation changes may improve protein quality, and provide needed macronutrients to food-aid receiving areas. Despite this, this research did not suggest that antinutritional factors affected iron bioavailability. In support of this finding, literature, and clinical studies presented here suggest that salivary proteins, including PRPs and cystatin, may serve as adaptive protective mechanisms against phytic acid and tannin consumption, and that further research may be warranted before further recommendations for their removal from food-aid are made.

Diss. (sammanfattning). Uppsala : Sveriges lantbruksuniversitet, 2005. / Härtill 5 uppsatser.

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Activation of the stress-responsive transcription factor NRF2 is the major line of defense to combat oxidative or electrophilic insults. Under basal conditions, NRF2 is continuously ubiquitylated by the KEAP1-CUL3-RBX1 E3 ubiquitin ligase complex and is targeted to the proteasome for degradation ( the canonical mechanism). However, the path from the CUL3 complex to ultimate proteasomal degradation was previously unknown. p97 is a ubiquitin-targeted ATP-dependent segregase that extracts ubiquitylated client proteins from membranes, protein complexes, or chromatin and has an essential role in autophagy and the ubiquitin proteasome system ( UPS). In this study, we show that p97 negatively regulates NRF2 through the canonical pathway by extracting ubiquitylated NRF2 from the KEAP1-CUL3 E3 complex, with the aid of the heterodimeric cofactor UFD1/NPL4 and the UBA-UBX containing protein UBXN7, for efficient proteasomal degradation. Given the role of NRF2 in chemoresistance and the surging interest in p97 inhibitors to treat cancers, our results indicate that dual p97/NRF2 inhibitors may offer a more potent and long-term avenue of p97-targeted treatment.