Hepatic CYP3A4 Enzyme Compensatively Maintains Endogenous Geranylgeranoic Acid Levels in MAOB-Knockout Human Hepatoma Cells.

Geranylgeranoic acid (GGA), developed as a preventive agent against second primary hepatoma, has been reported to be biosynthesized via the mevalonate pathway in human hepatoma-derived cells. Recently, we found that monoamine oxidase B (MAOB) catalyzed the oxidation of geranylgeraniol (GGOH) to produce geranylgeranial (GGal), a direct precursor of endogenous GGA in hepatoma cells, using tranylcypromine, an inhibitor of MAOs, and knockdown by MAOB siRNA. However, endogenous GGA level was unexpectedly unchanged in MAOB-knockout (KO) cells established using the CRISPR-Cas9 system, suggesting that some other latent metabolic pathways maintain endogenous GGA levels in the MAOB-KO cells. Here, we investigated the putative latent enzymes that oxidize GGOH in Hep3B/MAOB-KO cells. First, the broad-specific cytochrome P450 enzyme inhibitors decreased the amount of endogenous GGA in Hep3B/MAOB-KO cells in a dose-dependent manner. Second, among the eight members of cytochrome P450 superfamily that have been suggested to be involved in the oxidation of isoprenols and/or retinol in previous studies, only the CYP3A4 gene significantly upregulated its cellular mRNA level in Hep3B/MAOB-KO cells. Third, a commercially available recombinant human CYP3A4 enzyme was able to oxidize GGOH to GGal, and fourth, the knockdown of CYP3A4 by siRNA significantly reduced the amount of endogenous GGA in Hep3B/MAOB-KO cells. These results indicate that CYP3A4 can act as an alternative oxidase for GGOH when hepatic MAOB is deleted in the human hepatoma-derived cell line Hep3B, and that endogenous GGA levels are maintained by a multitude of enzymes.

Use of Parathyroid Hormone and Rehabilitation Reduces Subsequent Vertebral Body Fractures after Balloon Kyphoplasty.

STUDY DESIGN: Retrospective cohort study. PURPOSE: To evaluate the efficacy of our current prophylactic strategy by investigating the incidence of subsequent vertebral body fractures (SVBFs) following balloon kyphoplasty (BKP). OVERVIEW OF LITERATURE: Although extensive studies have investigated the risk factors for SVBFs after BKP, few have reported on postoperative therapies to prevent SVBFs and have evaluated their effectiveness. METHODS: This study enrolled 273 patients who underwent an initial BKP. To treat osteoporosis, parathyroid hormone (PTH) administration was started 1-2 weeks before BKP and continued for at least 6 months postoperatively. Corsets were applied for 3 months after the procedure. Rehabilitative interventions, including hip range-of-motion training, muscle strengthening exercises, and motion/posture instruction, were started from the preoperative assessment time point and resumed 3 hours postoperatively. Corsets were used in all patients. Therefore, no grouping based on corset use was performed. PTH was used in 180 patients, and they were divided into the following two groups: PTH user group and PTH nonuser group. Rehabilitative interventions were provided to all patients for a median duration of 17 days. Patients who underwent rehabilitative intervention for <17 and ≥17 days were included in the short-term and long-term intervention groups, respectively. The incidences of SVBFs for these four groups were compared. RESULTS: SVBF occurred in 29 patients (10.6%). The SVBF incidence among patients who were prescribed all three prophylactic measures was 6.2%. The PTH user group had a significantly lower incidence of distant vertebral body fractures as compared to the PTH nonuser group. The long-term rehabilitation group had a significantly lower incidence of SVBFs and adjacent vertebral body fractures within 50 postoperative days than the short-term group. CONCLUSIONS: A 17-day or longer rehabilitative intervention may lower the risk of early adjacent vertebral body fractures, and the use of PTH may reduce the risk of distant vertebral body fractures.

Age-Dependent Decrease in Hepatic Geranylgeranoic Acid Content in C3H/HeN Mice and Its Oral Supplementation Prevents Spontaneous Hepatoma.

Geranylgeranoic acid (GGA) has been developed as a preventive agent against second primary hepatoma. Recently, GGA was reported to induce cell death in human hepatoma cells via TLR4-mediated pyroptosis. We have reported that GGA is enzymatically biosynthesized from mevalonic acid in human hepatoma-derived cells and that endogenous GGA is found in most organs of rats. In addition, we found that upregulation of endogenous GGA levels by zaragozic acid A (ZAA) induced cell death in human hepatoma-derived cells. Therefore, we investigated the age-related changes in hepatic GGA and the possibility of suppressing hepatocarcinogenesis by GGA supplementation using male C3H/HeN mice that spontaneously develop hepatoma. We measured endogenous GGA and mRNA of monoamine oxidase (BMAOB), a key enzyme of GGA biosynthesis, in the liver of male C3H/HeN mice aged 6-93 weeks. We also tried suppressing spontaneous hepatocarcinogenesis by a single administration of GGA to C3H/HeN mice. Hepatic GGA content and Maob mRNA expression level age-dependently decreased in male C3H/HeN mice; some of which produced spontaneous hepatoma in 2 years. A single oral administration of GGA at 11 months of age significantly prevented hepatoma in terms of the number and weight of tumors per mouse at 24 months. Oral supplementation with GGA or geranylgeraniol significantly increased endogenous hepatic GGA contents dose-dependently; and ZAA dramatically upregulated hepatic GGA. In this study; we found an age-dependent decrease in hepatic endogenous GGA in male C3H/HeN mice and efficient prevention of spontaneous hepatoma by a single administration of GGA at 11 months of age.

Hepatic monoamine oxidase B is involved in endogenous geranylgeranoic acid synthesis in mammalian liver cells.

Geranylgeranoic acid (GGA) originally was identified in some animals and has been developed as an agent for preventing second primary hepatoma. We previously have also identified GGA as an acyclic diterpenoid in some medicinal herbs. Recently, we reported that in human hepatoma-derived HuH-7 cells, GGA is metabolically labeled from 13C-mevalonate. Several cell-free experiments have demonstrated that GGA is synthesized through geranylgeranial by oxygen-dependent oxidation of geranylgeraniol (GGOH), but the exact biochemical events giving rise to GGA in hepatoma cells remain unclear. Monoamine oxidase B (MOAB) has been suggested to be involved in GGOH oxidation. Here, using two human hepatoma cell lines, we investigated whether MAOB contributes to GGA biosynthesis. Using either HuH-7 cell lysates or recombinant human MAOB, we found that: 1) the MAO inhibitor tranylcypromine dose-dependently downregulates endogenous GGA levels in HuH-7 cells; and 2) siRNA-mediated MAOB silencing reduces intracellular GGA levels in HuH-7 and Hep3B cells. Unexpectedly, however, CRISPR/Cas9-generated MAOB-KO human hepatoma Hep3B cells had GGA levels similar to those in MAOB-WT cells. A sensitivity of GGA levels to siRNA-mediated MAOB downregulation was recovered when the MAOB-KO cells were transfected with a MAOB-expression plasmid, suggesting that MAOB is the enzyme primarily responsible for GGOH oxidation and that some other latent metabolic pathways may maintain endogenous GGA levels in the MAOB-KO hepatoma cells. Along with the previous findings, these results provide critical insights into the biological roles of human MAOB and provide evidence that hepatic MAOB is involved in endogenous GGA biosynthesis via GGOH oxidation.

Engineering pH-responsive switching of donor-π-acceptor chromophore alignments along a peptide nanotube scaffold.

A cyclic tri-ß-peptide cyclo(ß-Ala-ß-Ala-ß-Lys) having diethylaminonaphthalimide at the ß-Lys side chain (CP3Npi) self-assembled into a peptide nanotube in a solution of HFIP and water. CD spectra of the CP3Npi nanotubes show a negative Cotton effect at 441 nm and a positive Cotton effect at 393 nm, indicating that D-π-A naphthalimide chromophores are aligned in a left-handed chiral way along the nanotube. The CP3Npi nanotubes bear positive charges under acidic conditions retaining the nanotube structure but pH-responsive switching of D-π-A naphthalimide alignments along the nanotube between a left-handed chiral and random arrangement was observed. The peptide nanotube is a stable scaffold for attaining pH-responsive alignment switching of side-chain chromophores.

Endocannabinoid Signaling from 2-Arachidonoylglycerol to CB1 Cannabinoid Receptor Facilitates Reward-based Learning of Motor Sequence.

The endocannabinoid system modulates synaptic transmission, controls neuronal excitability, and is involved in various brain functions including learning and memory. 2-arachidonoylglycerol, a major endocannabinoid produced by diacylglycerol lipase-α (DGLα), is released from postsynaptic neurons, retrogradely activates presynaptic CB1 cannabinoid receptors, and induces short-term or long-term synaptic plasticity. To examine whether and how the endocannabinoid system contributes to reward-based learning of a motor sequence, we subjected male CB1-knockout (KO) and DGLα-KO mice to three types of operant lever-press tasks. First, we trained mice to press one of three levers labeled A, B, and C for a food reward (one-lever task). Second, we trained mice to press the three levers in the order of A, B, and C (three-lever task). Third, the order of the levers was reversed to C, B, and A (reverse three-lever task). We found that CB1-KO mice and DGLα-KO mice exhibited essentially the same deficits in the operant lever-press tasks. In the one-lever task, both strains of knockout mice showed a slower rate of learning to press a lever for food. In the three-lever task, both strains of knockout mice showed a slower rate of learning of the motor sequence. In the reverse three-lever task, both strains of knockout mice needed more lever presses for reversal learning. These results suggest that the endocannabinoid system facilitates reward-based learning of a motor sequence by conferring the flexibility with which animals can switch between strategies.

Unequivocal evidence for endogenous geranylgeranoic acid biosynthesized from mevalonate in mammalian cells.

Geranylgeranoic acid (GGA) has been reported to induce autophagic cell death via upregulation of lipid-induced unfolded protein response in several human hepatoma-derived cell lines, and its 4,5-didehydro derivative has been developed as a preventive agent against second primary hepatoma in clinical trials. We have previously reported that GGA is a natural diterpenoid synthesized in several medicinal herbs. Here, we provide unequivocal evidence for de novo GGA biosynthesis in mammals. First, with normal male Wistar rats, the levels of GGA in liver were found to be far greater than those in other organs analyzed. Second, we demonstrated the metabolic GGA labeling from the 13C-labeled mevalonolactone in the human hepatoma-derived cell line, HuH-7. Isotopomer spectral analysis revealed that approximately 80% of the cellular GGA was newly synthesized from mevalonate (MVA) in 12 h and the acid picked up preexisting farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP), suggesting that GGA is derived from FPP and GGPP through the MVA pathway. Third, zaragozic acid A, a squalene synthase inhibitor, induced dose-dependent upregulation of endogenous GGA content in HuH-7 cells and their concomitant cell death. These results strongly suggest that a cancer-preventive GGA is biosynthesized via the MVA pathway in mammals.

Chiral and random arrangements of flavin chromophores along cyclic peptide nanotubes on gold influencing differently on surface potential and piezoelectricity.

Two kinds of peptide nanotubes are prepared from cyclo(ß-Asp(flavin)-ß-alanine-ß-alanine) (C3FAA) and cyclo(ß-Asp(flavin)-ethylenediamine-succinic acid) (C3FES). The flavin chromophores are protruding on the C3FAA and C3FES peptide nanotube surfaces in random and chiral ways, respectively. The surface potentials of the C3FAA nanotube bundles on a gold substrate become larger than the C3FES nanotube bundles of the corresponding thicknesses. The converse piezoelectric coefficients are as small as less than 1 pm V-1. The peptide nanotube bundles are subjected to a thermal anneal treatment which raises up all the surface potentials and also the converse piezoelectricity of the C3FES nanotube bundles of 3 pm V-1. The macrodipole of the C3FAA nanotube and the chiral arrangement of the flavin groups in the C3FES nanotube are considered to contribute influentially to the surface potential and the piezoelectricity, respectively.

Piezoelectric property of bundled peptide nanotubes stapled by bis-cyclic-ß-peptide.

Cyclic tetra-ß-peptides (CP4s) and a bis-CP4 were synthesized to prepare peptide nanotubes (PNTs) by molecular stacking of cyclic peptides. The addition of bis-CP4 to the PNT preparation afforded PNT bundles increasing the direct and converse piezoelectiric coefficients, which is ascribable to bis-CP4 stapling PNTs into the parallel alignment of PNT dipoles.

Two one-dimensional arrays of naphthyl and anthryl groups along peptide nanotubes prepared from cyclic peptides comprising α- and ß-amino acids.

A novel cyclic hexapeptide composed of l-α-naphthylalanine, d-α-anthrylalanine, and four ß-alanines (CP6) is synthesized and its molecular assembly into peptide nanotubes (PNTs) and the electronic properties arising from one-dimensional arrays of aromatic groups along the PNTs were investigated. CP6 with a combination of l- and d-α-amino acids is designed to self-assemble into PNTs with them stacking on top of each other under the constraint of maximizing the number of intermolecular hydrogen bonds between the cyclic peptides. Upon PNT formation, the respective side chains of l- and d-α-amino acids are aligned in line along the PNTs. The topological arrangement of the anthryl groups being in close proximity in the CP6 PNT is supported by higher photo-excited energy transfer, appearance of the induced Cotton effects, and the promoted photo-dimerization reaction upon PNT formation. AFM observations reveal that PNT bundles with diameters 5-15 nm are dielectric microcrystals having a piezoelectric coefficient of 2-6 pC N-1. Kelvin force microscopy observations show the generation of surface potentials over 100 mV owing to the one-dimensional array of the anthryl groups along PNTs. Incorporation of α-amino acids with opposite chirality into cyclic ß-peptides is therefore an effective molecular design for the nano-architecture of PNTs displaying one-dimensional arrays of chromophores along PNTs.

CD28 co-stimulation is dispensable for the steady state homeostasis of intestinal regulatory T cells.

It is well-established that CD28 co-stimulation is required for the development and the proliferation of thymus-derived regulatory T cells (tTregs). Meanwhile, the role of CD28 co-stimulation in the homeostasis of peripherally derived Tregs (pTregs) remains unclear. To clarify this issue, we analyzed Tregs in small and large intestines (SI and LI), the principle sites of pTreg development. Interestingly, and different from in the thymus, Tregs were abundant in the intestines of CD28-/- mice, and most of them were phenotypically pTregs. We showed that CD28-/- naive T cells differentiated into pTregs in the LI after oral exposure to antigens and that CD28-/- pTregs in the LI had the same highly proliferative activity as CD28+/- cells. CD28-/- pTregs acquired these Treg-specific features at transcriptional and epigenetics levels. On the other hand, some immune suppressive molecules were down-regulated in CD28-/- pTregs. Correspondingly, the suppressive activity of CD28-/- pTregs was weaker than CD28+/+ cells. These results indicate that the homeostasis of pTregs in the intestines is maintained even in the absence of CD28, whereas CD28 is required for the maximal suppressive activity of intestinal pTregs.

Peptide nanotube aligning side chains onto one side.

A novel pseudo cyclic penta-ß-peptide composed of a ß-naphthylalanine, two ß-alanines, and a sequence of ethylenediamine-succinic acid (CP5ES) is synthesized and investigated on peptide nanotube (PNT) formation. When the PNT is formed with the maximum number of intermolecular hydrogen bonds between the cyclic peptides, the sequence enables the alignment of the side chains, naphthyl groups, on one side of the PNT. Microscopic and spectroscopic observations of CP5ES crystals reveal that CP5ES forms rod- or needle-shaped molecular assemblies showing exciton coupling of the Cotton effect and predominant monomer emission, which are different from a reference cyclic tri-ß-peptide composed of a ß-naphthylalanine and two ß-alanines. Insertion of a sequence of ethylenediamine-succinic acid into ß-amino acids in the cyclic skeleton is therefore suggested to be effective to make the side chains aligning on one side of the PNT. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Genetic screening for regulators of Prz1, a transcriptional factor acting downstream of calcineurin in fission yeast.

Calcineurin phosphatase plays crucial roles in a wide variety of cell types and organisms. Dephosphorylation of the nuclear factor of activated T-cell (NFAT) family of transcriptional factors by calcineurin is essential for activating immune-responsive genes in mammals. NFAT activity is also regulated by diverse signaling pathways, which affect NFAT kinases and nuclear partner proteins. In fission yeast, calcineurin dephosphorylates and activates Prz1, a C2H2-type zinc finger transcriptional factor. Calcineurin-Prz1 signaling regulates the expression of the Pmc1 Ca(2+) pump. Prz1-overexpressing cells showed extremely slow growth and high transcriptional activity of Prz1 in the absence of stimulation. Here, we isolated seven genes as dosage-dependent suppressors of this slow growth phenotype. These seven genes encode Rad24, Rad25, Pka1, Msn5 (SPAC328.01c), Pac1, Ape2, and Tfs1. All of them decreased the high transcriptional activity caused by Prz1 overexpression. Overexpression of Pka1, Rad24, and Rad25 also repressed the Ca(2+)-induced transcriptional activity in cells with Prz1 expressed at wild-type levels. Knock-out of rad24 or rad25 significantly enhanced the transcriptional activity of Prz1, whereas knock-out or mutation of other genes did not enhance the activity. The 14-3-3 proteins, Rad24 and Rad25, bound Prz1 and the Rad24-binding site located at residues 421-426 of Prz1. In msn5 deletion mutants, GFP-Prz1 localized at nucleus in the absence of Ca(2+) stimulation, suggesting that Msn5 functions as an exportin for Prz1. In summary, our data suggest that Rad24 and Rad25 negatively regulate Prz1 and that Pka1, Msn5, Pac1, Tfs1, and Ape2 also regulate Prz1.