<i>In vitro</i> inhibition and enhancement of liver microsomal S-777469 metabolism by long-chain fatty acids and serum albumin: insight into <i>in vitro</i> and <i>in vivo</i> discrepancy of metabolite formation in humans

<p>1. It was previously demonstrated that 10% of S-777469, a cannabinoid receptor 2 selective agonist, is metabolized to its carboxylic acid metabolite (S-777469 5-carboxylic acid, 5-CA) in humans <i>in vivo</i>, while the formation of 5-CA is extremely low in human cryopreserved hepatocytes and liver microsomes (HLMs). In this study, factors causing the different metabolite formation rates of S-777469 <i>in vitro</i> and <i>in vivo</i> were investigated.</p> <p>2. Formation of 5-CA and S-777469 5-hydroxymethyl (5-HM), a precursor metabolite of 5-CA, was catalyzed by CYP2C9. Arachidonic acid, α-linolenic acid, oleic acid and myristic acid, which have been reported to exist in liver microsomes, inhibited S-777469 oxidation by CYP2C9, but serum albumin enhanced this reactions.</p> <p>3. The IC₅₀ values of these fatty acids for 5-CA formation from 5-HM were lower than those of 5-HM formation from S-777469. Serum albumin extensively enhanced 5-CA formation from 5-HM in comparison to 5-HM formation from S-777469.</p> <p>4. CYP2C9 was the enzyme responsible for S-777469 oxidation in human livers. The suppressive effects of several fatty acids and enhancing action of serum albumin <i>in vitro</i> are likely to be the causal factors for the apparently different rates of <i>in vitro</i> and <i>in vivo</i> metabolite formation of S-777469.</p

Linear Bis(perfluoroalkyl) Complexes of Nickel Bipyridine

Three new complexes were prepared: [(dtbpy)­Ni­(CF₃)₂] (<b>1</b>), [(dtbpy)­Ni­(CF₂CF₃)₂] (<b>2</b>), and [(dtbpy)­Ni­(CH₃)₂] (<b>3</b>) (dtbpy = 4,4′-di-<i>tert</i>-butyl-2,2′-bipyridine). Remarkable differences in the structure, electronics, reactivity, and absorption of visible light for the alkyl versus perfluoroalkyl complexes were observed and are detailed in this report

The pre-OR region of PrP^ScΔOR is PK-resistant.

<p>(A) The brain homogenates of terminally ill wild-type and tg(PrPΔOR)/<i>Prnp^0/0</i> mice were treated with PNGase F after digestion with PK, and subjected to immunoblotting with M-20 anti-PrP antibodies. The deglycosylated PK-resistant band of PrP^ScΔOR was higher in molecular size than that of full-length PrP^Sc. Arrows indicates PK-resistant deglycosylated PrPs. (B) The brain homogenates from terminally ill wild-type and tg(PrPΔOR)/<i>Prnp^0/0</i> mice were digested with PK, and subjected to immunoblotting with N-terminus-specific IBL-N anti-PrP antibody. The IBL-N antibodies recognized the PK-resistant PrPs from PrP^ScΔOR but not from full-length PrP^Sc.</p

Lysine residues are important for the pre-OR residues 23–31 to form a PK-resistant structure in prion-infected N2a cells.

<p>(A) Amino acid sequences of the pre-OR residues 23–31 in moPrP(3F4) Δ32–88, moPrP(3F4) Δ32–88(3K3A) and moPrP(3F4) Δ32–88(2P2A). Bold residues indicate substituted alanine residues. (B) Western blotting of N2aC24L1-3 cells transfected with control pcDNA3.1(+) and expression vectors encoding moPrP(3F4), moPrP(3F4) Δ32–88, moPrP(3F4) Δ32–88(3K3A) and moPrP(3F4) Δ32–88(2P2A) using 3F4 anti-PrP antibodies. The cell lysates were treated with PK at 5 µg/ml. All mutant proteins were converted into PK-resistant isoforms in N2aC24L1-3 cells. The PK treatment revealed doublet non-glycosylated and mono-glycosylated bands in moPrP(3F4)^ScΔ32–88 (arrows), indicating that the pre-OR region of some moPrP(3F4)^ScΔ32–88 molecules is PK-resistant. Similar doublet bands were observed in moPrP(3F4)^ScΔ32–88(2P2A) (arrows). However, moPrP(3F4)^ScΔ32–88(3K3A) gave rise to doublet bands with the upper band migrating very closely to the lower band (arrowheads). (C) Since substitution of proline residues into alanine residues disrupted the IBL-N epitope, the PK-resistant pre-OR residues in moPrP(3F4)^ScΔ32–88(2P2A) failed to be visualized by IBL-N anti-PrP antibodies.</p

Overexpression of PrPΔOR in the brains and spinal cords of tg(PrPΔOR)/<i>Prnp^0/0</i> mice.

<p>The brain and spinal cord homogenates from tg(PrPΔOR)/<i>Prnp^0/0</i> mice and wild-type mice were subjected to Western blotting with SAF61 or SAF32 anti-PrP antibodies. The expression of β-actin was detected in these homogenates as an internal control.</p

PK-resistant pre-OR residues 23–31 of PrP^ScΔ32–88 generated in prion-infected N2a cells.

<p>(A) Schematic diagrams of moPrP(3F4) and moPrP(3F4) Δ32–88. Arabic numbers represent the codon numbers. SP, signal peptide; OR, octapeptide repeat; GPI, GPI anchor signal; α, α-helix; β, β-strand. (B, C) Western blotting of N2aC24L1-3 cells transfected with control pcDNA3.1(+), pcDNA3.1-moPrP(3F4), and pcDNA3.1-moPrP(3F4) Δ32–88 using 3F4 (B) or IBL-N anti-PrP antibodies (C). The cell lysates were treated with PK at 5 µg/ml and then subjected to Western blotting. Both moPrP(3F4) and moPrP(3F4) Δ32–88 were converted to the PK-resistant isoforms, moPrP^Sc(3F4) and moPrP^Sc(3F4) Δ32–88, respectively. However, IBL-N anti-PrP antibody reacted only with the PK-resistant fragments of moPrP^Sc(3F4) Δ32–88.</p

PK-resistant PrP accumulated in the brains and spinal cords of terminally ill tg(PrPΔOR)/<i>Prnp^0/0</i> mice.

<p>Immunoblots of the two PK-treated individual brains (A) and spinal cords (B) from terminally ill wild-type and tg(PrPΔOR)/<i>Prnp^0/0</i> mice using SAF61 anti-PrP antibodies.</p

Prion titers in the brains and spinal cords of terminally ill tg(PrPΔOR)/<i>Prnp^0/0</i> and wild-type mice inoculated with RML prions.

<p>Prion titers in the brains and spinal cords of terminally ill tg(PrPΔOR)/<i>Prnp^0/0</i> and wild-type mice inoculated with RML prions.</p

Astrogliosis in the brains and cervical cords of tg(PrPΔOR)/<i>Prnp^0/0</i> mice infected with RML prions.

<p>The cerebral cortices (A) and cervical cords (B) of uninfected or terminally ill wild-type and tg(PrPΔOR)/<i>Prnp^0/0</i> mice were immunohistochemically stained with anti-GFAP antibodies. The signals were milder in the brains of terminally ill tg(PrPΔOR)/<i>Prnp^0/0</i> mice, compared to those in control wild-type mice. No decrease in the signals was observed in the cervical cords of terminally ill tg(PrPΔOR)/<i>Prnp^0/0</i> mice. Immunoblots of the homogenates of brains (C) and spinal cords (D) of uninfected and terminally ill wild-type and tg(PrPΔOR)/<i>Prnp^0/0</i> mice using anti-GFAP antibodies are shown. Terminally ill tg(PrPΔOR)/<i>Prnp^0/0</i> mice expressed GFAP in their brains less than control wild-type mice. No reduction in the GFAP expression was detected in the spinal cords of terminally ill tg(PrPΔOR)/<i>Prnp^0/0</i> mice.</p

Mouse-Hamster Chimeric Prion Protein (PrP) Devoid of N-Terminal Residues 23-88 Restores Susceptibility to 22L Prions, but Not to RML Prions in PrP-Knockout Mice

<div><p>Prion infection induces conformational conversion of the normal prion protein PrP^C, into the pathogenic isoform PrP^Sc, in prion diseases. It has been shown that PrP-knockout (<i>Prnp^0/0</i>) mice transgenically reconstituted with a mouse-hamster chimeric PrP lacking N-terminal residues 23-88, or Tg(MHM2Δ23-88)/<i>Prnp^0/0</i> mice, neither developed the disease nor accumulated MHM2^ScΔ23-88 in their brains after inoculation with RML prions. In contrast, RML-inoculated Tg(MHM2Δ23-88)/<i>Prnp^0/+</i> mice developed the disease with abundant accumulation of MHM2^ScΔ23-88 in their brains. These results indicate that MHM2Δ23-88 itself might either lose or greatly reduce the converting capacity to MHM2^ScΔ23-88, and that the co-expressing wild-type PrP^C can stimulate the conversion of MHM2Δ23-88 to MHM2^ScΔ23-88 <i>in trans</i>. In the present study, we confirmed that Tg(MHM2Δ23-88)/<i>Prnp^0/0</i> mice remained resistant to RML prions for up to 730 days after inoculation. However, we found that Tg(MHM2Δ23-88)/<i>Prnp^0/0</i> mice were susceptible to 22L prions, developing the disease with prolonged incubation times and accumulating MHM2^ScΔ23-88 in their brains. We also found accelerated conversion of MHM2Δ23-88 into MHM2^ScΔ23-88 in the brains of RML- and 22L-inoculated Tg(MHM2Δ23-88)/<i>Prnp^0/+</i> mice. However, wild-type PrP^Sc accumulated less in the brains of these inoculated Tg(MHM2Δ23-88)/<i>Prnp^0/+</i> mice, compared with RML- and 22L-inoculated <i>Prnp^0/+</i> mice. These results show that MHM2Δ23-88 itself can convert into MHM2^ScΔ23-88 without the help of the <i>trans</i>-acting PrP^C, and that, irrespective of prion strains inoculated, the co-expressing wild-type PrP^C stimulates the conversion of MHM2Δ23-88 into MHM2^ScΔ23-88, but to the contrary, the co-expressing MHM2Δ23-88 disturbs the conversion of wild-type PrP^C into PrP^Sc.</p></div