Considerable evidence indicates that NO biology involves a family of NO-related molecules and that S-nitrosothiols (SNOs) are central to signal transduction and host defence. It is unknown, however, how cells switch off the signals or protect themselves from the SNOs produced for defence purposes. Here we have purified a single activity from Escherichia coli, Saccharomyces cerevisiae and mouse macrophages that metabolizes S-nitrosoglutathione (GSNO), and show that it is the glutathione-dependent formaldehyde dehydrogenase. Although the enzyme is highly specific for GSNO, it controls intracellular levels of both GSNO and S-nitrosylated proteins. Such 'GSNO reductase' activity is widely distributed in mammals. Deleting the reductase gene in yeast and mice abolishes the GSNO-consuming activity, and increases the cellular quantity of both GSNO and protein SNO. Furthermore, mutant yeast cells show increased susceptibility to a nitrosative challenge, whereas their resistance to oxidative stress is unimpaired. We conclude that GSNO reductase is evolutionarily conserved from bacteria to humans, is critical for SNO homeostasis, and protects against nitrosative stress.

The current perspective of NO biology is formulated predominantly from studies of NO synthesis. The role of S-nitrosothiol (SNO) formation and turnover in governing NO-related bioactivity remains uncertain. We generated mice with a targeted gene deletion of S-nitrosoglutathione reductase (GSNOR), and show that they exhibit substantial increases in whole-cell S-nitrosylation, tissue damage, and mortality following endotoxic or bacterial challenge. Further, GSNOR(-/-) mice have increased basal levels of SNOs in red blood cells and are hypotensive under anesthesia. Thus, SNOs regulate innate immune and vascular function, and are cleared actively to ameliorate nitrosative stress. Nitrosylation of cysteine thiols is a critical mechanism of NO function in both health and disease.

Only a few intracellular S-nitrosylated proteins have been identified, and it is unknown if protein S-nitrosylation/denitrosylation is a component of signal transduction cascades. Caspase-3 zymogens were found to be S-nitrosylated on their catalytic-site cysteine in unstimulated human cell lines and denitrosylated upon activation of the Fas apoptotic pathway. Decreased caspase-3 S-nitrosylation was associated with an increase in intracellular caspase activity. Fas therefore activates caspase-3 not only by inducing the cleavage of the caspase zymogen to its active subunits, but also by stimulating the denitrosylation of its active-site thiol. Protein S-nitrosylation/denitrosylation can thus serve as a regulatory process in signal transduction pathways.

Orphan G-protein-coupled receptors are a large class of receptors whose cognate ligands are unknown. SP9155 (also referred to as AQ27 and GPR103) is an orphan G-protein-coupled receptor originally cloned from a human brain cDNA library. SP9155 was found to be predominantly expressed in brain, heart, kidney, retina, and testis. Phylogenetic analysis shows that SP9155 shares high homology with Orexin, NPFF, and cholecystokinin (CCK) receptors, but identification of the endogenous ligand for SP9155 has not been reported. In this study, we have used a novel method to predict peptides from genome data bases. From these predicted peptides, a novel RF-amide peptide, P52 was shown to selectively activate SP9155-transfected cells. We subsequently cloned the precursor gene of the P52 ligand and characterized the activity of other possible peptides encoded by the precursor. This revealed an extended peptide, P518, which exhibited high affinity for SP9155 (EC 50 ‫؍‬ 7 nM). mRNA expression analysis revealed that the peptide P518 precursor gene is predominantly expressed in various brain regions, coronary arteries, thyroid and parathyroid glands, large intestine, colon, bladder, testes, and prostate. These results indicate the existence of a novel RF-amide neuroendocrine peptide system, and suggest that SP9155 is likely the relevant G-protein-coupled receptor for this peptide.G-protein coupled receptors (GPCRs) 1 are members of a large protein family that share a common structural motif of seven transmembrane domains (1, 2). GPCRs mediate a variety of physiological functions by interacting with extracellular ligands including small chemical molecules, peptides, and proteins, then transmitting the signal to intracellular second messengers via G proteins (1, 2). In recent years, the primary sequence of many GPCRs have been identified through searching the human genome sequence data base. Whereas these GPCRs have common structural motifs including seven transmembrane domains, often their cognate ligands and biological function are unknown (3-5). To understand the biological functions of these so called orphan receptors, and to enable identification of pharmacological agents active at these receptors, it is helpful to first identify their endogenous cognate ligands (3-5). Currently there are two main approaches generally used to identify orphan receptor ligands. One common approach has been to screen the orphan GPCRs against a collection of known or putative GPCR ligands. This approach has been used successfully to identify several ligand receptor pairings, including MCH, NMU, and P2Y13 etc. (6 -8). However, because ligand collections are somewhat limited, they often do not contain the appropriate ligand for the orphan receptor of interest. Another approach involves purification of the ligands from animal tissue extracts. This approach has also been successful in a number of cases including OFQ/Nociceptin, MCH, Orexin, and P2Y12 etc. (9 -13). However, purification of ligands from animal tissue extracts is labor-intensive and is c...

Cereal Chem. 74(1):63-71The starch of wheat (Triticum aestivum L.) flour affects food product quality due to the temperature-dependent interactions of starch with water during gelatinization, pasting, and gelation. The objective of this study was to determine the fundamental basis of variation in gelatinization, pasting, and gelation of prime starch derived from seven different wheat cultivars: Kanto 107, which is a partial waxy mutant line, and six near-isogenic lines (NILs) differing in hardness. Complete pasting curves with extended 16-min hold at 93°C were obtained using the Rapid Visco Analyser (RVA). Apparent amylose content ranged from 17.5 to 23.5%; total amylose content ranged from 22.8 to 28.2%. Starches exhibited significant variation in onset of gelatinization. However, none of the parameters measured consistently correlated with onset or other RVA curve parameters that preceded peak paste viscosity. Peak paste viscosity varied from 190 to 323 RVA units (RVU). Higher peak, greater breakdown, lower final viscosity, negative setback, and less total setback