A novel calreticulin-related molecule that interacts with bacteria and enhances host resistance against bacterial infection in black rockfish, Sebastes schlegeli.

Calreticulin (CRT) is a highly conserved and multi-functional protein with diverse localizations. CRT has lectin-like properties and possesses important immunological activities in mammalian. In teleost, very limited studies on CRT immunologic function have been documented. In the present study, a CRT homologue (SsCRT) was cloned, identified and characterized from black rockfish, Sebastes schlegeli, an important aquaculture species in East Asia. The full length of SsCRT cDNA is 2180 bp and encoded a polypeptide of 425 amino acids. SsCRT contains a signal peptide, three distinct structural and functional domains (N-, P- and C-domains), and an endoplasmic reticulum (ER) retrieval signal sequence (KDEL). The deduced amino acid sequence of SsCRT shares 89-92% overall sequence identities with the CRT proteins of several fish species. SsCRT was distributed ubiquitously in all the detected tissues and was highly expressed in the spleen, muscle and liver. After the infection of fish extracellular bacterial pathogen Vibrio anguillarum and intracellular bacterial pathogen Edwardsiella tarda, the mRNA transcripts of SsCRT in spleen, liver, and head kidney were significantly up-regulated. The expression patterns were time-dependent and tissue-dependent. Recombinant SsCRT (rSsCRT) exhibited apparent binding activities against different bacteria and PAMPs. In vivo studies showed that the expressions of multiple immune-related genes such as TNF13B, IL-1ß, IL-8, SAA, Hsp70, and ISG15 in head kidney were significantly enhanced when black rockfish were treated with rSsCRT. Furthermore, rSsCRT reduced pathogen dissemination and replication in fish kidney and spleen. These results indicated that SsCRT served as an immune receptor to recognize and eliminate the invading pathogens, which played a vital role in the immune response of Sebastes schlegeli. These findings provide new insights into understanding the roles of CRT proteins in immune response and pathogen infection in teleost.

Inhibition potential of UDP-glucuronosyltransferases (Ugts) 1A isoforms by the analogue of resveratrol, bakuchiol.

Bakuchiol is a promising anti-tumor candidate with resveratrol-like structure. The present study aims to evaluate the inhibition potential of bakuchiol towards UDP-glucuronosyltransferases (UGT) 1A isoforms. An in vitro incubation system using 4-methylumbelliferone (4-MU) glucuronidation was used to evaluate the inhibition capability of bakuchiol towards UGT1A1, 1A3, 1A6, 1A7, 1A8, 1A9 and 1A10. The glucuronidation of trifluoperazine (TFP) was employed as the probe reaction to determine bakuchiol's inhibition towards UGT1A4. At 1 microM and 10 microM of bakuchiol, no or weak inhibition was observed for all the tested UGT1A isoforms. At 100 microM of bakuchiol, the activity of UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9 and 1A10 was inhibited by -46.2%, 74.7%, 17.8%, 98.7%, 70.4%, 99.2%, 75.8%, and 93.3%, respectively. Further inhibition kinetic behaviour was determined for UGT1A6, 1A8, and 1A10. Both Dixon plot and Lineweaver-Burk plot showed the noncompetitive inhibition of bakuchiol towards all these three UGT isoforms. The inhibition kinetic parameters (Ki) were calculated to be 5.3, 1.8, and 92.6 microM for UGT1A6, 1A8, and 1A10, respectively. In combination with the in vivo exposure of bakuchiol, the high possibility of in vivo inhibition of UGT1A6 and 1A8 was predicted. However, relatively low possibility of in vivo inhibition towards UGT1A10 was predicted due to lower in vivo concentration of bakuchiol than its inhibition parameter (Ki). All these information will be helpful for the R&D of bakuchiol as a promising anti-tumor drug.