Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that regulates genes involved in xenobiotic metabolism, cellular proliferation, and differentiation. Numerous xenobiotic and biological compounds are known to interact with AhR, but it remains an orphan receptor, because its physiological ligand is unknown. We identified AhR ligands in human urine using a yeast AhR signaling assay and then characterized their properties. Two ligands, indirubin and indigo, were both present at average concentrations of ϳ0.2 nM in the urine of normal donors. Indirubin was also detected in fetal bovine serum and contributed half of the total AhR ligand activity. The activities of indirubin and indigo were comparable with or more potent than that of the archetypal ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin, in yeast AhR activation assays. We suggest that the endogenous levels and potencies of indirubin and indigo are such that they activate AhR-mediated signaling mechanisms in vivo. AhR,1 also called the dioxin receptor, is a ligand-activated transcription factor that is present in most cell and tissue types of the body (1). AhR-mediated signaling is required for potent xenobiotic ligands such as TCDD and polychlorinated biphenyls to produce toxic responses (2, 3). Toxic effects that are linked to xenobiotic AhR ligand exposures in animals include cancers, reproductive impairment, endometriosis, birth defects, and immunological impairment (4 -6). The toxic potential of xenobiotic AhR ligands is currently a major concern for regulatory agencies that are responsible for protecting public and environmental health. Although numerous xenobiotic ligands for AhR have been identified, the AhR is considered to be an orphan receptor, because its physiological ligand(s) and its function are not known. Tryptophan and other indole-containing compounds (7-9), bilirubin (10), 7-ketocholesterol (11), lipoxin A4 (12), flavones, and related compounds (13) interact with AhR to produce activation or inhibition of signal transduction. In general, the low levels, lack of potency, and restricted distribution of these compounds make them unlikely candidates as major regulators of AhR signaling in most tissue types. We reasoned that human urine might be a good place to search for endogenous AhR ligands, and we developed a methodology to detect and isolate such compounds. EXPERIMENTAL PROCEDURESMaterials-Blue rayon was kindly provided by Dr. Hayatsu (Okayama University, Okayama City, Japan). General chemicals, essentially analytical grade, were purchased from Wako (Kyoto, Japan). TCDD was purchased from CIL (Andover, MA, USA). Indigo and ␤-glucuronidase were purchased from Sigma. Indirubin was synthesized as described in Hoessel et al. (14). Indigo and indirubin were further purified by HPLC before use.Yeast Assay for AhR Ligand Activity-The assay procedure was essentially as described by Miller (15). The yeast strain YCM3 was grown overnight at 30°C in synthetic glucose medium lacking tryptophan. Test chemicals (dissolved in Me 2 SO...

The ubiquitin-proteasome system is essential for intracellular protein degradation, but an extracellular role of this system has not been known until now. We have previously reported that the proteasome is secreted into the surrounding seawater from sperm of the ascidian (Urochordata) Halocynthia roretzi on sperm activation, and that the sperm proteasome plays a key role in fertilization. Here, we show that a 70-kDa component (HrVC70) of the vitelline coat is the physiological substrate for the ubiquitin-proteasome system during fertilization of H. roretzi. A cDNA clone encoding the HrVC70 precursor (HrVC120) was isolated, and a homology search revealed that HrVC120 contains 13 epidermal growth factor-like repeats and a mammalian zona pellucida glycoprotein-homologous domain. HrVC70 functions as a sperm receptor. We demonstrate that HrVC70 is ubiquitinated both in vitro and in vivo. The immunocytochemical localization of multiubiquitin chains in the vitelline coat and the inhibitory effect of monoclonal antibodies against the multiubiquitin chains on fertilization strongly support the role of the ubiquitin-proteasome system in ascidian fertilization. Taken together, these results indicate that the ubiquitin-proteasome system is responsible for extracellular degradation of the sperm receptor HrVC70 and, consequently, for sperm penetration of the vitelline coat during fertilization.fertilization ͉ vitelline coat ͉ ubiquitin F ertilization is a key event in sexual reproduction, creating a new individual with novel genomic information. In animal reproduction, species-specific binding of sperm to the proteinaceous egg coat, called the vitelline coat in marine invertebrates or the zona pellucida in mammals, is particularly important for successful fertilization. Because the egg coat is a potential barrier to sperm-egg fusion, sperm must use a lytic agent (lysin) to penetrate it (1, 2). In mammals, it has long been believed that the sperm acrosomal trypsin-like protease acrosin is a zona lysin, which digests zona pellucida proteins to enable sperm to penetrate through the zona pellucida (3). However, recent studies by using acrosin-knockout mice revealed that acrosin is not essential for fertilization or sperm penetration through the zona pellucida (4). Rather, it is currently thought that acrosin is involved in the dispersal of acrosomal proteins during acrosome reaction (5) and that a sperm protease(s) other than acrosin is the actual zona-lysin in mammalian fertilization (6).Ascidians (Urochordata) occupy a phylogenetic position between invertebrates and segmented vertebrates. They are hermaphrodites that usually release sperm and eggs simultaneously during the spawning season. Several ascidians, including Ciona intestinalis (7) and Halocynthia roretzi (8), strictly prohibit selffertilization. Because self-nonself recognition in fertilization is accomplished by interaction between the sperm and vitelline coat (7,8), the sperm-lysin system seems to be triggered after the sperm recognizes the vitelline coat as nonself...

Empathy cultivates deeper interpersonal relationships and is important for socialization. However, frequent exposure to emotionally-demanding situations may put people at risk for burnout. Burnout has become a pervasive problem among medical professionals because occupational burnout may be highly sensitive to empathy levels. To better understand empathy-induced burnout among medical professionals, exploring the relationship between burnout severity and strength of empathy-related brain activity may be key. However, to our knowledge, this relationship has not yet been explored. We studied the relationship between self-reported burnout severity scores and psychological measures of empathic disposition, emotional dissonance and alexithymia in medical professionals to test two contradictory hypotheses: Burnout is explained by (1) ‘compassion fatigue' that is, individuals become emotionally over involved; and (2) ‘emotional dissonance' that is, a gap between felt and expressed emotion, together with reduced emotional regulation. Then, we tested whether increased or decreased empathy-related brain activity measured by fMRI was associated with burnout severity scores and psychological measures. The results showed that burnout severity of medical professionals is explained by ‘reduced' empathy-related brain activity. Moreover, this reduced brain activity is correlated with stronger emotional dissonance and alexithymia scores and also greater empathic disposition. We speculate that reduced emotion recognition (that is, alexithymia) might potentially link with stronger emotional dissonance and greater burnout severity alongside empathy-related brain activity. In this view, greater empathic disposition in individuals with higher burnout levels might be due to greater difficulty identifying their own emotional reactions. Our study sheds new light on the ability to predict empathy-induced burnout.

Ascidians are hermaphrodites releasing sperm and eggs simultaneously, but many species are self-sterile because of a self͞nonself-recognition system in sperm-egg interaction. Here, we show that a 70-kDa vitelline coat protein, HrVC70, consisting of 12 epidermal growth factor-like repeats, plays a key role in self͞nonself recognition during ascidian fertilization. We discovered that the amount of HrVC70 of the self-sterile mature oocytes is markedly higher than that of the self-fertile immature oocytes and that the selfsterile mature oocytes become self-fertile by acid treatment, which is able to release the HrVC70 from isolated vitelline coats. In addition, fertilization is strongly inhibited by the pretreatment of sperm with HrVC70 from a different individual, but not from the same individual, and the number of nonself sperm bound to HrVC70-agarose was significantly higher than that of self-sperm. A sequence analysis of HrVC70 disclosed that several amino acid residues in a restricted region are substituted at an individual level, with no identical sequences among the 10 individuals tested. Furthermore, genomic DNA analysis revealed that the epidermal growth factor-like domains correspond to the exons, and each intron is highly conserved among even-and odd-numbered introns, suggesting that multiple gene duplications or amplification of this region might have taken place during evolution. It was also found that diversity in cDNA sequences is derived from genomic DNA polymorphism probably elicited by crossing over and specific nucleotide substitutions. These results indicate that HrVC70 is a candidate allogeneic recognition molecule in the gamete interaction of the ascidian Halocynthia roretzi.