In vitro antioxidant and antimutagenic activity of dietary chlorophyll derivatives was assessed. Antioxidant activity was determined by the ability of each compound to scavenge the long-lived free radicals 1,1-diphenyl-2-picrylhydrazyl (DPPH · ) and 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS ·+ ). Antimutagenic activity was assayed with a modified microscreen bacterial reverse mutagenicity assay using Salmonella typhimurium TA100 and benzo[a]pyrene as the tester strain and mutagen respectively. Derivatives of chlorophyll a were found to be more effective radical quenchers than those of chlorophyll b. Furthermore, metal-free derivatives such as chlorins, pheophytins, and pyropheophytins exhibited significantly lower antiradical capacity than metalloderivatives such as Mg-chlorophylls, Zn-pheophytins, Zn-pyropheophytins, Cu-pheophytin a, and Cu-chlorophyllins. Both metal-free and metallo-chlorophyll derivatives demonstrated similar dose-dependent inhibitory activity against B[a]P induced mutagenesis. These results demonstrate that dietary chlorophyll derivatives prevalent in both fresh and processed foods and dietary supplements have antioxidant and antimutagenic activities.

Phosphatidylinositol transfer proteins (PITPs) have been shown to play important roles in regulating a number of signal transduction pathways that couple to vesicle trafficking reactions, phosphoinositide-driven receptor-mediated signaling cascades, and development. While yeast and metazoan PITPs have been analyzed in some detail, plant PITPs remain entirely uncharacterized. We report the identification and characterization of two soybean proteins, Ssh1p and Ssh2p, whose structural genes were recovered on the basis of their abilities to rescue the viability of PITP-deficient Saccharomyces cerevisiae strains. We demonstrate that, while both Ssh1p and Ssh2p share approximately 25% primary sequence identity with yeast PITP, these proteins exhibit biochemical properties that diverge from those of the known PITPs. Ssh1p and Ssh2p represent high-affinity phosphoinositide binding proteins that are distinguished from each other both on the basis of their phospholipid binding specificities and by their substantially non-overlapping patterns of expression in the soybean plant. Finally, we show that Ssh1p is phosphorylated in response to various environmental stress conditions, including hyperosmotic stress. We suggest that Ssh1p may function as one component of a stress response pathway that serves to protect the adult plant from osmotic insult.

Although phosphatidylinositol transfer proteins (PITPs) are known to serve critical functions in regulating a varied array of signal transduction processes in animals and yeast, the discovery of a similar class of proteins in plants occurred only recently. Here, we report the participation of Ssh1p, a soybean PITP-like protein, in the early events of osmosensory signal transduction in plants, a function not attributed previously to animal or yeast PITPs. Exposure of plant tissues to hyperosmotic stress led to the rapid phosphorylation of Ssh1p, a modification that decreased its ability to associate with membranes. An osmotic stress-activated Ssh1p kinase activity was detected in several plant species by presenting recombinant Ssh1p as a substrate in in-gel kinase assays. Elements of a similar osmosensory signaling pathway also were conserved in yeast, an observation that facilitated the identification of soybean protein kinases SPK1 and SPK2 as stress-activated Ssh1p kinases. This study reveals the activation of SPK1 and/or SPK2 and the subsequent phosphorylation of Ssh1p as two early successive events in a hyperosmotic stress-induced signaling cascade in plants. Furthermore, Ssh1p is shown to enhance the activities of a plant phosphatidylinositol 3-kinase and phosphatidylinositol 4-kinase, an observation that suggests that the ultimate function of Ssh1p in cellular signaling is to alter the plant's capacity to synthesize phosphoinositides during periods of hyperosmotic stress. INTRODUCTIONPhosphatidylinositol transfer proteins (PITPs) were identified originally by their ability to serve as diffusible carriers of phosphatidylinositol (PtdIns) and to a lesser extent phosphatidylcholine (PtdCho) from one distinct membrane compartment to another by using an in vitro assay (Wirtz, 1991). In recent years, several intriguing and critical biological roles beyond the transfer of phospholipids have been attributed to yeast and animal PITPs. The yeast PITP (Sec14p) is an essential protein that is required for cells to properly execute the formation of secretory vesicles from the Golgi complex (Bankaitis et al., 1990). A considerable body of evidence suggests that Sec14p serves as a "molecular sensor" to monitor and regulate the levels of PtdIns, PtdCho, and potentially diacylglycerol in the Golgi complex of yeast (Skinner et al., 1995;Kearns et al., 1997). In addition, Sec14p has been implicated in modulating the activity of a PtdIns 4-kinase that regulates protein secretion (Hama et al., 1999).An essential role for PITPs also is observed in mammals and Drosophila, in which the loss of PITP function leads to specific neurodegenerative diseases (Hamilton et al., 1997;Milligan et al., 1997). At the cellular level, the mammalian PITP is known to be required for inositol lipid signaling, secretory vesicle formation from the trans -Golgi network, and the fusion of secretory vesicles to the plasma membrane (Hay and Martin, 1993;Cunningham et al., 1995;Kauffmann-Zeh et al., 1995). Although yeast and animal PITPs are very similar...

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Umami plays an important role in the flavor of many cheese varieties. The purpose of this study was to identify the compound(s) responsible for umami taste in Cheddar and Swiss cheeses. Four Cheddar and 4 Swiss cheeses (two with low umami intensity and two with high umami intensity from each type) were selected using a trained sensory panel. Monosodium glutamate (MSG), disodium 5'-inosine monophosphate (IMP), disodium 5'-guanosine monophosphate (GMP), sodium chloride, lactic acid, propionic acid, and succinic acid were quantified in the cheeses instrumentally. Taste thresholds (best estimate thresholds, BETs) were determined for each compound in water. Subsequently, a trained descriptive sensory analysis panel evaluated each compound in odor-free water across threshold concentrations to confirm that the thresholds were based on umami and not some other stimuli. Model system studies with trained panelists were then conducted with each compound individually or all compounds together. Comparison of analytical data and sensory thresholds indicated that IMP and GMP thresholds were 100-fold higher than their concentrations in cheese. All other compounds contributed some umami taste within their concentration range in umami cheeses. Sensory analysis of model cheeses revealed that glutamic acid played the largest role in umami taste of both Cheddar and Swiss cheeses while succinic and propionic acids contributed to umami taste in Swiss cheeses. Knowledge of the key compounds associated with umami taste in cheeses will aid in the identification of procedures to enhance formation of this taste in cheese.

The activity of chymosin, plasmin, and Lactococcus lactis enzymes (cell envelope proteinase, intracellular peptidases, and glycolytic enzymes) were determined after 5-min exposures to pressures up to 800 MPa. Plasmin was unaffected by any pressure treatment. Chymosin activity was unaffected up to 400 MPa and decreased at 500 to 800 MPa. Fifty percent of control chymosin activity remained after the 800 MPa treatment. The lactococcal cell envelope proteinase (CEP) and intracellular peptidase activities were monitored in cell extracts of pressure-treated cells. A pressure of 100 MPa increased the CEP activity, whereas 200 MPa had no effect. At 300 MPa, CEP activity was reduced, and 400 to 800 MPa inactivated the enzyme. X-Prolyl-dipeptidyl aminopeptidase was insensitive to 5-min pressure treatments of 100 to 300 MPa, but was inactivated at 400 to 800 MPa. Aminopeptidase N was unaffected by 100 and 200 MPa. However, 300 MPa significantly reduced its activity, and 400 to 800 MPa inactivated it. Aminopeptidase C activity increased with increasing pressures up to 700 MPa. High pressure did not affect aminopeptidase A activity at any level. Hydrolysis of Lys-Ala-p-NA doubled after 300-MPa exposure, and was eliminated at 400 to 800 MPa. Glycolytic enzyme activities of pressure-treated cells were evaluated collectively by determining the titratable acidity as lactic acid produced by cell extracts in the presence of glucose. The titratable acidities produced by the 100 and 200 MPa samples were slightly increased compared to the control. At 300 to 800 MPa, no significant acid production was observed. These data demonstrate that high pressure causes no effect, activation, or inactivation of proteolytic and glycolytic enzymes depending on the pressure level and enzyme. Pressure treatment of cheese may alter enzymes involved in ripening, and pressure-treating L. lactis may provide a means to generate attenuated starters with altered enzyme profiles.

Although the accumulation of the PLMT substrates phosphatidylmonomethylethanolamine and phosphatidyldimethylethanolamine was considerably elevated in the atplmt knock-out line, PtdCho levels remained normal, and no obvious differences were observed in plant morphology or development under standard growth conditions. However, because the metabolic routes through which PtdCho is synthesized in plants vary greatly among differing species, it is predicted that the degree with which PtdCho synthesis is dependent upon PLMT activities will also vary widely throughout the plant kingdom.

Five Lactobacillus strains of intestinal and food origins were grown in MRS broth or milk containing various concentrations of linoleic acid or conjugated linoleic acid (CLA). The fatty acids had bacteriostatic, bacteriocidal, or no effect depending on bacterial strain, fatty acid concentration, fatty acid type, and growth medium. Both fatty acids displayed dose-dependent inhibition. All strains were inhibited to a greater extent by the fatty acids in broth than in milk. The CLA isomer mixture was less inhibitory than linoleic acid. Lactobacillus reuteri ATCC 55739, a strain capable of isomerizing linoleic acid to CLA, was the most inhibited strain by the presence of linoleic acid in broth or milk. In contrast, a member of the same species, L. reuteri ATCC 23272, was the least inhibited strain by linoleic acid and CLA. All strains increased membrane linoleic acid or CLA levels when grown with exogenous fatty acid. Lactobacillus reuteri ATCC 55739 had substantial CLA in the membrane when the growth medium was supplemented with linoleic acid. No association between level of fatty acid incorporation into the membrane and inhibition by that fatty acid was observed.