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.