Ovine corticotropin-releasing hormone (oCRH) exerts an anxiogenic-like action in the goldfish, Carassius auratus.

Corticotropin-releasing hormone (CRH) is a member of the hypothalamic neuropeptide family that includes urocortins, urotensin I and sauvagine in vertebrates. CRH and urocortin act as anorexigenic factors for satiety regulation in rodents. In a goldfish model, intracerebroventricular (ICV) administration of ovine CRH (oCRH) affects not only food intake, but also locomotor activity. However, there is no information regarding the psychophysiological roles of CRH in goldfish. Therefore, we investigated the effect of oCRH on psychomotor activity in this species. ICV administration of synthetic oCRH at 20 pmol/g body weight (BW) enhanced locomotor activity. Since intact goldfish prefer the lower to the upper area of a tank, we developed a method for measuring the time taken for fish to move from the lower to the upper area. ICV administration of oCRH at 20 pmol/g BW and the central-type benzodiazepine receptor inverse agonist FG-7142 (an anxiogenic agent) at 1-4 pmol/g BW both increased the time taken to move from the lower to the upper area. This anxiogenic-like effect of oCRH was abolished by the CRH receptor antagonist α-helical CRH(9-41) (100 pmol/g BW). These results indicate that CRH can potently affect locomotor and psychomotor activities in goldfish.

ERp57 binds competitively to protein disulfide isomerase and calreticulin.

In this study, we screened for protein disulfide isomerase (PDI)-binding proteins in bovine liver microsomes under strict salt concentrations, using affinity column chromatography. One main band observed using SDS-PAGE was identified as ERp57 (one of the PDI family proteins) by LC-MS/MS analysis. The K(D) value of PDI binding to ERp57 was calculated as 5.46x10(-6)M with the BIACORE system. The interactions between PDI and ERp57 occurred specifically at their a and b domains, respectively. Interestingly, low concentrations of ERp57 enhanced the chaperone activity of PDI, while high concentrations interfered with chaperone activity. On the other hand, ERp57 did not affect the isomerase activity of PDI. Additionally, following pre-incubation of ERp57 with calreticulin (CRT), decreased interactions were observed between ERp57 and PDI, and vice versa. Based on the data, we propose that once ERp57 binds to PDI or CRT, the resultant complex inhibits further interactions. Therefore, ERp57 selectively forms a protein-folding complex with PDI or CRT in ER.

The chaperone activity of protein disulfide isomerase is affected by cyclophilin B and cyclosporin A in vitro.

To elucidate the function of protein disulfide isomerase (PDI), we screened for PDI-binding proteins in a bovine liver extract using affinity column chromatography. One of the binding proteins was identified by SDS-PAGE and N-terminal amino acid sequence analysis to be cyclophilin B (Cyp B). Use of the BIACORE system revealed that purified bovine Cyp B bound specifically to bovine PDI with a K(D) value of 1.19 x 10(-5) M. Interestingly, the binding affinity between PDI and Cyp B was strengthened by preincubation of the Cyp B with cyclosporin A (CsA), yielding a K(D) value of 3.67 x 10(-6) M. Although the interaction between PDI and Cyp B affected neither the isomerase activity of PDI nor the peptidyl-prolyl cis-trans isomerase activity of Cyp B, Cyp B increased the chaperone activity of PDI. However, the complex of Cyp B and CsA completely inhibited the chaperone activity of PDI. Thus, PDI and Cyp B appear to cooperate with each other to regulate the functional expression of proteins in vivo.