Vitamin K occurs in the natural world in several forms, including a plant form, phylloquinone (PK), and a bacterial form, menaquinones (MKs). In many species, including humans, PK is a minor constituent of hepatic vitamin K content, with most hepatic vitamin K content comprising long-chain MKs. Menaquinone-4 (MK-4) is ubiquitously present in extrahepatic tissues, with particularly high concentrations in the brain, kidney and pancreas of humans and rats. It has consistently been shown that PK is endogenously converted to MK-4 (refs 4-8). This occurs either directly within certain tissues or by interconversion to menadione (K(3)), followed by prenylation to MK-4 (refs 9-12). No previous study has sought to identify the human enzyme responsible for MK-4 biosynthesis. Previously we provided evidence for the conversion of PK and K(3) into MK-4 in mouse cerebra. However, the molecular mechanisms for these conversion reactions are unclear. Here we identify a human MK-4 biosynthetic enzyme. We screened the human genome database for prenylation enzymes and found UbiA prenyltransferase containing 1 (UBIAD1), a human homologue of Escherichia coli prenyltransferase menA. We found that short interfering RNA against the UBIAD1 gene inhibited the conversion of deuterium-labelled vitamin K derivatives into deuterium-labelled-MK-4 (MK-4-d(7)) in human cells. We confirmed that the UBIAD1 gene encodes an MK-4 biosynthetic enzyme through its expression and conversion of deuterium-labelled vitamin K derivatives into MK-4-d(7) in insect cells infected with UBIAD1 baculovirus. Converted MK-4-d(7) was chemically identified by (2)H-NMR analysis. MK-4 biosynthesis by UBIAD1 was not affected by the vitamin K antagonist warfarin. UBIAD1 was localized in endoplasmic reticulum and ubiquitously expressed in several tissues of mice. Our results show that UBIAD1 is a human MK-4 biosynthetic enzyme; this identification will permit more effective decisions to be made about vitamin K intake and bone health.

We examined the effect of 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand, on the intracellular free Ca 2؉ concentrations in HL-60 cells that express the cannabinoid CB2 receptor. We found that 2-arachidonoylglycerol induces a rapid transient increase in intracellular free Ca 2؉ concentrations in HL-60 cells. The response was affected by neither cyclooxygenase inhibitors nor lipoxygenase inhibitors, suggesting that arachidonic acid metabolites are not involved. Consistent with this notion, free arachidonic acid was devoid of any agonistic activity. Importantly, the Ca 2؉ transient induced by 2-arachidonoylglycerol was blocked by pretreatment of the cells with SR144528, a CB2 receptor-specific antagonist, but not with SR141716A, a CB1 receptor-specific antagonist, indicating the involvement of the CB2 receptor but not the CB1 receptor in this cellular response. G i or G o is also assumed to be involved, because pertussis toxin treatment of the cells abolished the response. We further examined the structure-activity relationship. We found that 2-arachidonoylglycerol is the most potent compound among a number of naturally occurring cannabimimetic molecules. Interestingly, anandamide and N-palmitoylethanolamine, other putative endogenous ligands, were found to be a weak partial agonist and an inactive ligand, respectively. These results strongly suggest that the CB2 receptor is originally a 2-arachidonoylglycerol receptor, and 2-arachidonoylglycerol is the intrinsic natural ligand for the CB2 receptor that is abundant in the immune system. ⌬ 9 -Tetrahydrocannabinol (⌬ 9 -THC), 1 a major psychoactive constituent of marijuana, is known to exert a variety of pharmacological effects on laboratory animals and humans. When administered orally or intravenously, ⌬ 9 -THC induces reduced spontaneous motor activity, analgesia, heightened sensory awareness, euphoria, and impairment of short-term memory (1). ⌬ 9 -THC is also known to exert profound effects on several biological systems other than the central nervous system; for example, ⌬ 9 -THC exhibits potent immunosuppressive activities (2-12). The mechanism underlying such diverse actions of ⌬ 9 -THC remained obscure until the late 1980s. -THC itself bind to a specific receptor site(s) thereby eliciting a variety of responses. To date, two types of cannabinoid receptors have been identified as follows: the CB1 receptor expressed primarily in the nervous system (14), and the CB2 receptor expressed mainly in the immune system (15). Both are seven transmembrane, G protein-coupled receptors, and they share 44% overall identity (68% identity for transmembrane domains) (15).The discovery of specific receptors for cannabinoids prompted the search for endogenous ligands. To date, two types of arachidonic acid-containing molecules, anandamide (16) and 2-arachidonoylglycerol (2-AG) (17, 18), have been reported as putative endogenous cannabinoid receptor ligands. Anandamide was isolated from porcine brain (16), and 2-AG was isolated from rat brain (17) and canine gut...

Several reports indicate an important role for vitamin K in bone health as well as blood coagulation. However, the current Adequate Intakes (AI) might not be sufficient for the maintenance of bone health. To obtain a closer estimate of dietary intake of phylloquinone (PK) and menaquinones (MKs), PK, MK-4 and MK-7 contents in food samples (58 food items) were determined by an improved high-performance liquid chromatography method. Next, we assessed dietary vitamin K intake in young women living in eastern Japan using vitamin K contents measured here and the Standard Tables of Food Composition in Japan. PK was widely distributed in green vegetables and algae, and high amounts were found in spinach and broccoli (raw, 498 and 307 g/100 g wet weight, respectively). Although MK-4 was widely distributed in animal products, overall MK-4 content was lower than PK. MK-7 was observed characteristically in fermented soybean products such as natto (939 g/100 g). The mean total vitamin K intake of all subjects (using data from this study and Japanese food composition tables) was about 230 g/d and 94% of participants met the AI of vitamin K for women aged 18-29 y in Japan, 60 g/d. The contributions of PK, MK-4 and MK-7 to total vitamin K intake were 67.7, 7.3 and 24.9%, respectively. PK from vegetables and algae and MK-7 from pulses (including fermented soybean foods) were the major contributors to the total vitamin K intake of young women living in eastern Japan.

An endogenous cannabimimetic molecule, 2-arachidonoylglycerol, induces a rapid, transient increase in intracellular free Ca 2؉ concentrations in NG108 -15 cells through a cannabinoid CB1 receptor-dependent mechanism. We examined the activities of 24 relevant compounds (2-arachidonoylglycerol, its structural analogues, and several synthetic cannabinoids). We found that 2-arachidonoylglycerol is the most potent compound examined so far: its activity was detectable from as low as 0.3 nM, and the maximal response induced by 2-arachidonoylglycerol exceeded the responses induced by others. Activities of HU-210 and CP55940, potent cannabinoid receptor agonists, were also detectable from as low as 0.3 nM, whereas the maximal responses induced by these compounds were low compared with 2-arachidonoylglycerol. Anandamide was also found to act as a partial agonist in this assay system. We confirmed that free arachidonic acid failed to elicit a response. Furthermore, we found that a metabolically stable ether-linked analogue of 2-arachidonoylglycerol possesses appreciable agonistic activity, although its activity was apparently lower than that of 2-arachidonoylglycerol. We also confirmed that pretreating cells with various cannabinoid receptor agonists nullified the response induced by 2-arachidonoylglycerol, whereas pretreating cells with other neurotransmitters or neuromodulators did not affect the response. These results strongly suggested that the cannabinoid CB1 receptor is originally a 2-arachidonoylglycerol receptor, and 2-arachidonoylglycerol is the intrinsic physiological ligand for the cannabinoid CB1 receptor.It is well known that ⌬ 9 -tetrahydrocannabinol (⌬ 9 -THC), 1 a psychoactive ingredient of marijuana, possesses a variety of pharmacological activities in vitro and in vivo (1), although, until recently, the mechanism of the action of ⌬ 9 -THC had long been unelucidated. In 1988, Devane et al. (2) provided evidence that a specific binding site(s) for cannabinoids is present in the brain. Soon after, Matsuda et al. (3) cloned a cDNA encoding a cannabinoid receptor (CB1) from a rat brain cDNA library. These findings raised the possibility that at least part of the action of ⌬ 9 -THC is mediated through such a specific receptor and prompted the search for endogenous cannabinoid receptor ligands in mammalian tissues.In 1992, Devane et al. (4) isolated N-arachidonoylethanolamine (anandamide) from porcine brain as the first endogenous cannabinoid receptor ligand. They demonstrated that anandamide exhibits several cannabimimetic activities in vitro and in vivo (4, 5). So far, a number of studies have been carried out on anandamide, and it has been assumed that anandamide is one of the important lipid mediators in the nervous system as well as in other systems (5). However, we (6, 7) and others (8 -11) have found that the levels of anandamide are very low in several mammalian tissues. In addition, the biosynthetic pathways for anandamide, either the N-acylphosphatidylethanolamine pathway (6,7,(11)(12)(13)(14) or t...

A series of 1-N-iminosugars were synthesized to supply the need for glycosidase inhibitors that are both highly potent and selective for β-glycosidases. Designed on the basis of the transition-state model of the β-glucosidase reaction, these iminosugar inhibitors differ from the currently available inhibitors in possessing a nitrogen atom at the anomeric position of the pyranose ring, thereby generating a positive charge on the anomeric position rather than on the ring oxygen of the sugar. Their syntheses, starting with a readily available carbohydrate derivative, involve (i) introduction of an amino functionality as an azido group, (ii) formation of a 1-N-iminopyranose ring with reductive amination, and (iii) stereoselective introduction of a hydroxymethyl or methyl group and were accomplished in a highly stereoselective and efficient manner. The inhibitory potencies of the 1-N-iminosugars were evaluated against several α- and β-glycosidases, and they were found to be extremely potent and highly specific against the corresponding β-glycosidases, with K i values in the nanomolar range.