Insertion of circularly permuted cyan fluorescent protein into the ligand-binding domain of inositol 1,4,5-trisphosphate receptor for enhanced FRET upon binding of fluorescent ligand.

Binding of fluorescent ligand (FL) to the cyan fluorescent protein (CFP)-coupled ligand-binding domain of the inositol 1,4,5-trisphosphate (IP3) receptor (CFP-LBP) produces fluorescence (Förster) resonance energy transfer (FRET). A competitive fluorescent ligand assay (CFLA), using the FRET signal from competition between FLs and IP3, can measure IP3 concentration. The FRET signal should be enhanced by attaching a FRET donor to an appropriate position. Herein, we inserted five different circularly permuted CFPs in the loop between the second and third α-helices to generate membrane-targeted fluorescent ligand-binding proteins (LBPs). Two such proteins, LBP-cpC157 and LBP-cpC173, localized at the plasma membrane, displayed FRET upon binding the high-affinity ligand fluorescent adenophostin A (F-ADA), and exhibited a decreased fluorescence emission ratio (480 nm / 535 nm) by 1.6- to 1.8-fold that of CFP-LBP. In addition, binding of a fluorescent low-affinity ligand (F-LL) also reduced the fluorescence ratio in a concentration-dependent manner, with EC50 values for LBP-cpC157 and LBP-cpC173 of 34.7 nM and 27.6 nM, respectively. These values are comparable to that with CFP-LBP (29.2 nM), indicating that insertion of cpC157 and cpC173 did not disrupt LBP structure and function. The effect of 100 nM F-LL on the decrease in fluorescence ratio was reversed upon addition of IP3, indicating binding competition between F-LL and IP3. We also constructed cytoplasmic fluorescent proteins cyLBP-cpC157 and cyLBP-cpC173, and bound them to DYK beads for imaging analyses. Application of F-ADA decreased the fluorescence ratio of the beads from the periphery to the center over 3 - 5 min. Application of F-LL also decreased the fluorescence ratio of cyLBP-cpC157 and cyLBP-cpC173 by 20-25%, and subsequent addition of IP3 recovered the fluorescence ratio in a concentration-dependent manner. The EC50 value and Hill coefficient obtained by curve fitting against the IP3-dependent recovery of fluorescence ratio can be used to estimate the IP3 concentration.

Randomized Trial of Vitamin D Supplementation to Prevent Seasonal Influenza and Upper Respiratory Infection in Patients With Inflammatory Bowel Disease.

BACKGROUND: We evaluated whether oral vitamin D supplementation during the winter and early spring reduces the incidence of influenza and upper respiratory infections in patients with inflammatory bowel disease (IBD). METHODS: A randomized, double-blind, controlled trial was conducted to compare the effects of vitamin D supplementation (500 IU/day) and a placebo. The primary outcome was the incidence of influenza; the secondary outcome was the incidence of upper respiratory infection. Prespecified subgroup analyses were performed according to 25-hydroxyvitamin D (25-OHD) levels (low <20 ng/mL or high ≥20 ng/mL) and whether ulcerative colitis (UC) or Crohn's disease (CD) was present. We also used the Lichtiger clinical activity index for patients with UC and the Crohn's Disease Activity Index (CDAI) for patients with CD before and after interventions. RESULTS: We included 223 patients with IBD and randomized them into 2 groups: vitamin D supplementation (n = 108) and placebo (n = 115). The incidence of influenza did not differ between the groups. However, the incidence of upper respiratory infection was significantly lower in the vitamin D group (relative risk [RR], 0.59; 95% confidence interval (CI), 0.35-0.98; P = 0.042). This effect was enhanced in the low 25-OHD level subgroup (RR, 0.36; 95% CI, 0.14-0.90; P = 0.02). With respect to adverse events, the Lichtiger clinical activity index score was significantly worse in the vitamin D group (P = 0.002) and remained significant only in the high 25-OHD level subgroup. CONCLUSIONS: Vitamin D supplementation may have a preventative effect against upper respiratory infection in patients with IBD but may worsen the symptoms of UC.

Intestinal transepithelial permeability of oxytocin into the blood is dependent on the receptor for advanced glycation end products in mice.

Plasma oxytocin (OT) originates from secretion from the pituitary gland into the circulation and from absorption of OT in mother's milk into the blood via intestinal permeability. However, the molecular mechanism underlying the absorption of OT remains unclear. Here, we report that plasma OT concentrations increased within 10 min after oral delivery in postnatal day 1-7 mice. However, in Receptors for Advanced Glycation End Products (RAGE) knockout mice after postnatal day 3, an identical OT increase was not observed. In adult mice, plasma OT was also increased in a RAGE-dependent manner after oral delivery or direct administration into the intestinal tract. Mass spectrometry evaluated that OT was absorbed intact. RAGE was abundant in the intestinal epithelial cells in both suckling pups and adults. These data highlight that OT is transmitted via a receptor-mediated process with RAGE and suggest that oral OT supplementation may be advantageous in OT drug development.

Effect of hydrocotarnine on cytochrome P450 and P-glycoprotein.

The injectable form of oxycodone contains hydrocotarnine that is supposed to potentiate the analgesic effect of oxycodone with unknown mechanism(s). In this study, the effects of hydrocotarnine on the cytochrome P450 (CYP) and P-glycoprotein (P-gp) were investigated. Hydrocotarnine did not induce a significant change in the metabolic activities of CYP2C9, 2C19, and 2E1 in an in vitro study using human CYP recombinants. Although weak inhibitory effects were observed on CYP3A4 and 2D6, these interactions did not seem to be clinically relevant. Hydrocotarnine also did not cause a significant change in the ATPase activity of human P-gp membranes, suggesting that it is not an inhibitor of P-gp. Furthermore, mice were intraperitoneally injected with hydrocotarnine for 14 days and the mRNA levels of major CYP isozymes and P-gp in the liver and small intestine were determined by real-time RT-PCR. As a result, none of the mRNAs investigated showed a significant change in their levels by hydrocotarnine treatment. In conclusion, it is unlikely that the potentiation of oxycodone effect by hydrocotarnine involves its effect on CYP and P-gp. The findings also demonstrate that hydrocotarnine is unlikely to cause drug interactions via CYP or P-gp.

Analysis of risk factors for hepatocellular carcinoma that is negative for hepatitis B surface antigen (HBsAg).

OBJECTIVE: To clarify risk factors for hepatocellular carcinoma (HCC) other than hepatitis B surface antigen (HBsAg). PATIENTS AND METHODS: We investigated serum HBV-DNA and other factors in 146 patients with liver cirrhosis (LC) or HCC who were HBsAg negative. We analyzed the clinical background of the patients, status of hepatitis B (HBV) viral markers and platelet count as well as the presence of an HBV-DNA fragment by PCR and elucidated risk factors for HCC generation using a logistic regression model. RESULTS: Among ten factors, we determined that four represented a significant risk for HBsAg negative HCC: male gender, total alcohol consumption, total cigarettes smoked, and the presence of an HBV-DNA fragment. Multivariate analysis showed that among the four factors, the HBV-DNA fragment was an independent factor associated with HCC. CONCLUSION: The presence of an HBV-DNA fragment irrespective of the status of antibodies to either HBsAg (anti-HBs) or hepatitis B core antigen (anti-HBc) is a pivotal factor associated with the development of HCC.