Effects of 6-O-α-maltosyl-ß cyclodextrin on lipid metabolism in Npc1-deficient Chinese hamster ovary cells.

Niemann-Pick disease Type C (NPC) is a lysosomal storage disorder caused by mutation of the NPC1/NPC2 genes, which ultimately results in the accumulation of unesterified cholesterol (UEC) in lysosomes, thereby inducing symptoms such as progressive neurodegeneration and hepatosplenomegaly. This study determines the effects of 6-O-α-maltosyl-ß cyclodextrin (Mal-ßCD) on lipid levels and synthesis in Npc1-deficient (Npc1-KO cells) and vehicle CHO cells. Compared to vehicle cells, Npc1-KO cells exhibited high level of UEC, and low levels of esterified cholesterols (ECs) and long-chain fatty acids (LCFAs). The difference in lipid levels between Npc1-KO and CHO cells was largely ameliorated by Mal-ßCD administration. Moreover, the effects of Mal-ßCD were reproduced in the lysosomes prepared from Npc1-KO cells. Stable isotope tracer analysis with extracellular addition of D4-deuterated palmitic acid (D4-PA) to Npc1-KO cells increased the synthesis of D4-deuterated LCFAs (D4-LCFAs) and D4-deuterated ECs (D4-ECs) in a Mal-ßCD-dependent manner. Simultaneous addition of D6-deuterated UEC (D6-UEC) and D4-PA promoted the Mal-ßCD-dependent synthesis of D6-/D4-ECs, consisting of D6-UEC and D4-PA, D4-deuterated stearic acid, or D4-deuterated myristic acid, in Npc1-KO cells. These results suggest that Mal-ßCD helps to maintain normal lipid metabolism by restoring balance among UEC, ECs, and LCFAs through acting on behalf of NPC1 in Npc1-KO cells and may therefore be useful in designing effective therapies for NPC.

Inhibition of Escherichia coli Lipoprotein Diacylglyceryl Transferase Is Insensitive to Resistance Caused by Deletion of Braun's Lipoprotein.

Lipoprotein diacylglyceryl transferase (Lgt) catalyzes the first step in the biogenesis of Gram-negative bacterial lipoproteins which play crucial roles in bacterial growth and pathogenesis. We demonstrate that Lgt depletion in a clinical uropathogenic Escherichia coli strain leads to permeabilization of the outer membrane and increased sensitivity to serum killing and antibiotics. Importantly, we identify G2824 as the first-described Lgt inhibitor that potently inhibits Lgt biochemical activity in vitro and is bactericidal against wild-type Acinetobacter baumannii and E. coli strains. While deletion of a gene encoding a major outer membrane lipoprotein, lpp, leads to rescue of bacterial growth after genetic depletion or pharmacologic inhibition of the downstream type II signal peptidase, LspA, no such rescue of growth is detected after Lgt depletion or treatment with G2824. Inhibition of Lgt does not lead to significant accumulation of peptidoglycan-linked Lpp in the inner membrane. Our data validate Lgt as a novel antibacterial target and suggest that, unlike downstream steps in lipoprotein biosynthesis and transport, inhibition of Lgt may not be sensitive to one of the most common resistance mechanisms that invalidate inhibitors of bacterial lipoprotein biosynthesis and transport. IMPORTANCE As the emerging threat of multidrug-resistant (MDR) bacteria continues to increase, no new classes of antibiotics have been discovered in the last 50 years. While previous attempts to inhibit the lipoprotein biosynthetic (LspA) or transport (LolCDE) pathways have been made, most efforts have been hindered by the emergence of a common mechanism leading to resistance, namely, the deletion of the gene encoding a major Gram-negative outer membrane lipoprotein lpp. Our unexpected finding that inhibition of Lgt is not susceptible to lpp deletion-mediated resistance uncovers the complexity of bacterial lipoprotein biogenesis and the corresponding enzymes involved in this essential outer membrane biogenesis pathway and potentially points to new antibacterial targets in this pathway.

Cytotoxic Effects of Water-Soluble Extracts of Coarse and Fine Atmospheric Particulate Matter on Mast Cell Lines.

Fine particulate matter (PM2.5) pollution causes serious health disorders, because PM2.5 becomes deposited in the tracheobronchial and alveoli regions. In the extrathoracic region, there are more deposits of coarse particulate matter than fine particulates. As adverse health issues caused by coarse particulates have not been well investigated, this study examined the cytotoxicity of water-soluble extracts of both fine (0.05-3 µm, PM0.05-3) and coarse (> 3 µm, PM>3) particulates collected from April 2016 to March 2019 in Fukuoka, Japan. Also evaluated were concentrations of NH4+ and SO42-, multi-components of well-known secondary generation substances. The findings revealed that PM>3 showed stronger cytotoxic effects on mast cell lines than PM0.05-3. Cytotoxic effects were observed at concentrations of over 15 mM of (NH4)2SO4 and over 30 mM of NH4Cl. In contrast, Na2SO4 caused few cytotoxic effects up to a concentration of 50 mM. The causative substances for this cytotoxicity may not have been NH4+ and SO42- because their PM>3 concentrations indicating the largest cytotoxic effects were 1 and 0.4 mM, respectively. The cytotoxicities of PM>3 and PM0.05-3 were the highest in the first half of FY2016. These cytotoxicities seem to be due to cross-border pollution, although this pollution has been declining in recent years. An increasing trend of cytotoxicity was observed in the second half of FY2018. This study showed that cytotoxicity and particulate concentrations are not always correlated. Thus, we should focus not only on the quantity of atmospheric particulate matter, but also on its quality.

A New Bitterness Evaluation Index Obtained Using the Taste Sensor for 48 Active Pharmaceutical Ingredients of Pediatric Medicines.

The aim of this study was to evaluate bitterness by using "CCDP; Change in concentration-dependent potential" considering dose-dependency of active pharmaceutical ingredients (APIs) as new and useful bitterness evaluation index compared with bitter sensor output value which is conventional bitterness evaluation index for 48 pediatric medicines from the recent edition of the WHO model list of essential medicines for children (7th edn, 2019). Solutions (0.01, 0.03, 0.1 mM) of the compounds were evaluated by an artificial taste sensor using membranes sensitive to bitterness. The dose-response slope of the sensor outputs was defined as CCDP. On the basis of principal component analysis of CCDPs, chlorpromazine hydrochloride, amitriptyline hydrochloride, propranolol hydrochloride, primaquine phosphate and haloperidol were predicted to express the strongest levels of basic bitterness, surpassing that of quinine hydrochloride. Correlation analysis (Fisher's exact tests and multiple regression analysis) was performed to determine the relation between CCDPs and various physicochemical properties participated in hydrophilicity and hydrophobicity. It is revealed that contribution physicochemical factors are different by individual basic bitterness sensor (AC0, AN0 or BT0), and this result becomes the criterion of the sensor choice to evaluate basic bitterness intensity using basic bitterness sensors. Hydrophobic and hydrophilic interactions could be simulated by ligand docking modeling for haloperidol, miconazole and quinine hydrochloride. The pharmaceutical products need a bitterness evaluation in consideration of concentration-dependency to vary in a dose depending on a patient individual. Thus, it was concluded that CCDP correlated to hydrophilicity and hydrophobicity is useful as a bitterness evaluation index of APIs in pediatric medicines.

Antimicrobial Activities of LL-37 Fragment Mutant-Poly (Lactic-Co-Glycolic) Acid Conjugate against Staphylococcus aureus, Escherichia coli, and Candida albicans.

Various peptides and their derivatives have been reported to exhibit antimicrobial activities. Although these activities have been examined against microorganisms, novel methods have recently emerged for conjugation of the biomaterials to improve their activities. Here, we prepared CKR12-PLGA, in which CKR12 (a mutated fragment of human cathelicidin peptide, LL-37) was conjugated with poly (lactic-co-glycolic) acid (PLGA), and compared the antimicrobial and antifungal activities of the conjugated peptide with those of FK13 (a small fragment of LL-37) and CKR12 alone. The prepared CKR12-PLGA was characterized by dynamic light scattering and measurement of the zeta potential, critical micellar concentration, and antimicrobial activities of the fragments and conjugate. Although CKR12 showed higher antibacterial activities than FK13 against Staphylococcus aureus and Escherichia coli, the antifungal activity of CKR12 was lower than that of FK13. CKR12-PLGA showed higher antibacterial activities against S. aureus and E. coli and higher antifungal activity against Candida albicans compared to those of FK13. Additionally, CKR12-PLGA showed no hemolytic activity in erythrocytes, and scanning and transmission electron microscopy suggested that CKR12-PLGA killed and disrupted the surface structure of microbial cells. Conjugation of antimicrobial peptide fragment analogues was a successful approach for obtaining increased microbial activity with minimized cytotoxicity.

Bitterness-Suppressing Effect of Umami Dipeptides and Their Constituent Amino Acids on Diphenhydramine: Evaluation by Gustatory Sensation and Taste Sensor Testing.

Diphenhydramine, a sedating antihistamine, is an agonist of human bitter taste receptor 14 (hTAS2R14). Diphenhydramine hydrochloride (DPH) was used as a model bitter medicine to evaluate whether the umami dipeptides (Glu-Glu and Asp-Asp) and their constituent amino acids (Glu, Asp) could suppress its bitterness intensity, as measured by human gustatory sensation testing and using the artificial taste sensor. Various concentrated (0.001-5.0 mM) Glu-Glu, Asp-Asp, Glu and Asp significantly suppressed the taste sensor output of 0.5 mM DPH solution in a dose-dependent manner. The effect of umami dipeptides and their constituent amino acids was tending to be ranked as follows, Asp-Asp > Glu-Glu >> Gly-Gly, and Asp > Glu >> Gly (control) respectively. Whereas human bitterness intensity of 0.5 mM DPH solution with various concentrated (0.5, 1.0, 1.5 mM) Glu-Glu, Asp-Asp, Glu and Asp all significantly reduced bitterness intensity of 0.5 mM DPH solution even though no statistical difference was observed among four substances. The taste sensor outputs and the human gustatory sensation test results showed a significant correlation. A surface plasmon resonance study using hTAS2R14 protein and these substances suggested that the affinity of Glu-Glu, Asp-Asp, Glu and Asp for hTAS2R14 protein was greater than that of Gly-Gly or Gly. The results of docking-simulation studies involving DPH, Glu-Glu and Asp-Asp with hTAS2R14, suggested that DPH is able to bind to a space near the binding position of Glu-Glu and Asp-Asp. In conclusion, the umami dipeptides Glu-Glu and Asp-Asp, and their constituent amino acids, can all efficiently suppress the bitterness of DPH.

In Vitro and In Vivo Evaluation of 6-O-α-Maltosyl-ß-Cyclodextrin as a Potential Therapeutic Agent Against Niemann-Pick Disease Type C.

Niemann-Pick disease Type C (NPC) is a rare lysosomal storage disease characterized by the dysfunction of intracellular cholesterol trafficking with progressive neurodegeneration and hepatomegaly. We evaluated the potential of 6-O-α-maltosyl-ß-cyclodextrin (G2-ß-CD) as a drug candidate against NPC. The physicochemical properties of G2-ß-CD as an injectable agent were assessed, and molecular interactions between G2-ß-CD and free cholesterol were studied by solubility analysis and two-dimensional proton nuclear magnetic resonance spectroscopy. The efficacy of G2-ß-CD against NPC was evaluated using Npc1 deficient Chinese hamster ovary (CHO) cells and Npc1 deficient mice. G2-ß-CD in aqueous solution showed relatively low viscosity and surface activity; characteristics suitable for developing injectable formulations. G2-ß-CD formed higher-order inclusion complexes with free cholesterol. G2-ß-CD attenuated dysfunction of intercellular cholesterol trafficking and lysosome volume in Npc1 deficient CHO cells in a concentration dependent manner. Weekly subcutaneous injections of G2-ß-CD (2.9 mmol/kg) ameliorated abnormal cholesterol metabolism, hepatocytomegaly, and elevated serum transaminases in Npc1 deficient mice. In addition, a single cerebroventricular injection of G2-ß-CD (21.4 µmol/kg) prevented Purkinje cell loss in the cerebellum, body weight loss, and motor dysfunction in Npc1 deficient mice. In summary, G2-ß-CD possesses characteristics favorable for injectable formulations and has therapeutic potential against in vitro and in vivo NPC models.

Identification of Candidate Target Cyp Genes for microRNAs Whose Expression Is Altered by PCN and TCPOBOP, Representative Ligands of PXR and CAR.

MicroRNAs (miRNAs) are small non-coding RNAs that are involved in mRNA post-transcriptional regulation. The deregulation of miRNAs affects the expression of drug-metabolizing enzymes, drug transporters, and nuclear receptors, all of which are important in regulating drug metabolism. miRNA expression can be altered by several endogenous or exogenous agents, such as steroid hormones, carcinogens, and therapeutic drugs. However, it is unclear whether hepatic miRNA expression is regulated by nuclear receptors, such as pregnane X receptor (PXR) and constitutive androstane receptor (CAR), which are indispensable for the expression of the CYPs. Here we investigated the effects of the mouse PXR and CAR ligands pregnenolone-16α-carbonitrile (PCN) and 1,4-bis[(3,5-dichloropyridin-2-yl)oxy]benzene (TCPOBOP) on hepatic miRNA expression in mice. We found that the expression of 9 miRNAs was increased (>2-fold) and of 4 miRNAs was decreased (>50%) in response to PCN, while TCPOBOP treatment led to the up-regulation of 8 miRNAs and down-regulation of 6 miRNAs. Using several miRNA target prediction algorithms, we found that the predicted target genes included several lesser known Cyp genes (Cyp1a1, Cyp1b1, Cyp2b10, Cyp2c38, Cyp2u1, Cyp4a12a/b, Cyp4v3, Cyp17a1, Cyp39a1, and Cyp51). We analyzed the expression of these genes in response to PCN and TCPOBOP and found changes in their mRNA levels, some of which were negatively correlated with the expression of their corresponding miRNAs, suggesting that miRNAs may play a role in regulating Cyp enzyme expression. Further studies will be required to fully elucidate the miRNA regulatory mechanisms that contribute to modulating CYP expression.

The anchor-away technique: rapid, conditional establishment of yeast mutant phenotypes.

The anchor-away (AA) technique depletes the nucleus of Saccharomyces cerevisiae of a protein of interest (the target) by conditional tethering to an abundant cytoplasmic protein (the anchor) by appropriate gene tagging and rapamycin-dependent heterodimerization. Taking advantage of the massive flow of ribosomal proteins through the nucleus during maturation, a protein of the large subunit was chosen as the anchor. Addition of rapamycin, due to formation of the ternary complex, composed of the anchor, rapamycin, and the target, then results in the rapid depletion of the target from the nucleus. All 43 tested genes displayed on rapamycin plates the expected defective growth phenotype. In addition, when examined functionally, specific mutant phenotypes were obtained within minutes. These are genes involved in protein import, RNA export, transcription, sister chromatid cohesion, and gene silencing. The AA technique is a powerful tool for nuclear biology to dissect the function of individual or gene pairs in synthetic, lethal situations.

Selective association between nucleosomes with identical DNA sequences.

Self-assembly is the autonomous organization of constituents into higher order structures or assemblages and is a fundamental mechanism in biological systems. There has been an unfounded idea that self-assembly may be used in the sensing and pairing of homologous chromosomes or chromatin, including meiotic chromosome pairing, polytene chromosome formation in Diptera and transvection. Recent studies proved that double-stranded DNA molecules have a sequence-sensing property and can self-assemble, which may play a role in the above phenomena. However, to explain these processes in terms of self-assembly, it first must be proved that nucleosomes retain a DNA sequence-sensing property and can self-assemble. Here, using atomic force microscopy (AFM)-based analyses and a quantitative interaction assay, we show that nucleosomes with identical DNA sequences preferentially associate with each other in the presence of Mg(2+) ions. Using Xenopus borealis 5S rDNA nucleosome-positioning sequence and 601 and 603 sequences, homomeric or heteromeric octa- or tetranucleosomes were reconstituted in vitro and induced to form weak intracondensates by MgCl(2). AFM clearly showed that DNA sequence-based selective association occurs between nucleosomes with identical DNA sequences. Selective association was also detected between mononucleosomes. We propose that nucleosome self-assembly and DNA self-assembly constitute the mechanism underlying sensing and pairing of homologous chromosomes or chromatin.

Structural and functional characteristics of S-like ribonucleases from carnivorous plants.

Although the S-like ribonucleases (RNases) share sequence homology with the S-RNases involved in the self-incompatibility mechanism in plants, they are not associated with this mechanism. They usually function in stress responses in non-carnivorous plants and in carnivory in carnivorous plants. In this study, we clarified the structures of the S-like RNases of Aldrovanda vesiculosa, Nepenthes bicalcarata and Sarracenia leucophylla, and compared them with those of other plants. At ten positions, amino acid residues are conserved or almost conserved only for carnivorous plants (six in total). In contrast, two positions are specific to non-carnivorous plants. A phylogenetic analysis revealed that the S-like RNases of the carnivorous plants form a group beyond the phylogenetic relationships of the plants. We also prepared and characterized recombinant S-like RNases of Dionaea muscipula, Cephalotus follicularis, A. vesiculosa, N. bicalcarata and S. leucophylla, and RNS1 of Arabidopsis thaliana. The recombinant carnivorous plant enzymes showed optimum activities at about pH 4.0. Generally, poly(C) was digested less efficiently than poly(A), poly(I) and poly(U). The kinetic parameters of the recombinant D. muscipula enzyme (DM-I) and A. thaliana enzyme RNS1 were similar. The k cat/K m of recombinant RNS1 was the highest among the enzymes, followed closely by that of recombinant DM-I. On the other hand, the k cat/K m of the recombinant S. leucophylla enzyme was the lowest, and was ~1/30 of that for recombinant RNS1. The magnitudes of the k cat/K m values or k cat values for carnivorous plant S-like RNases seem to correlate negatively with the dependency on symbionts for prey digestion.

Most methylation-susceptible DNA sequences in human embryonic stem cells undergo a change in conformation or flexibility upon methylation.

DNA methylation in eukaryotes occurs on the cytosine bases in CG, CHG, and CHH (where H indicates non-G nucleotides) contexts and provides an important epigenetic mark in various biological processes. However, the structural and physical properties of methylated DNA are poorly understood. Using nondenaturing polyacrylamide gel electrophoresis, we performed a systematic study of the influence of DNA methylation on the conformation and physical properties of DNA for all CG, CHG, and CHH contexts. In the CG context, methylated multimers of the CG/CG-containing unit fragment migrated in gels slightly faster than their unmethylated counterparts. In the CHG context, both homo- and hemimethylation caused retarded migration of multimers of the CAG/CTG-containing fragment. In the CHH context, methylation caused or enhanced retarded migration of the multimers of CAA/TTG-, CAT/ATG-, CAC/GTG-, CTA/TAG-, or CTT/AAG-containing fragments. These results suggest that methylation increases DNA rigidity in the CG context and introduces distortions into several CHG and CHH sequences. More interestingly, we found that nearly all of the methylation repertoires in the CHG context and 98% of those in the CHH context in human embryonic stem cells were species that undergo conformational changes upon methylation. Similarly, most of the methylation repertoires in the Arabidopsis CHG and CHH contexts were sequences with methylation-induced distortion. We hypothesize that the methylation-induced properties or conformational changes in DNA may facilitate nucleosome formation, which provides the essential mechanism for alterations of chromatin density.

S-like ribonuclease gene expression in carnivorous plants.

Functions of S-like ribonucleases (RNases) differ considerably from those of S-RNases that function in self-incompatibility. Expression of S-like RNases is usually induced by low nutrition, vermin damage or senescence. However, interestingly, an Australian carnivorous plant Drosera adelae (a sundew), which traps prey with a sticky digestive liquid, abundantly secretes an S-like RNase DA-I in the digestive liquid even in ordinary states. Here, using D. adelae, Dionaea muscipula (Venus flytrap) and Cephalotus follicularis (Australian pitcher plant), we show that carnivorous plants use S-like RNases for carnivory: the gene da-I encoding DA-I and its ortholog cf-I of C. follicularis are highly expressed and constitutively active in each trap/digestion organ, while the ortholog dm-I of D. muscipula becomes highly active after trapping insects. The da-I promoter is unmethylated only in its trap/digestion organ, glandular tentacles (which comprise a small percentage of the weight of the whole plant), but methylated in other organs, which explains the glandular tentacles-specific expression of the gene and indicates a very rare gene regulation system. In contrast, the promoters of dm-I, which shows induced expression, and cf-I, which has constitutive expression, were not methylated in any organs examined. Thus, it seems that the regulatory mechanisms of the da-I, dm-I and cf-I genes differ from each other and do not correlate with the phylogenetic relationship. The current study suggests that under environmental pressure in specific habitats carnivorous plants have managed to evolve their S-like RNase genes to function in carnivory.

[Effects of Coarse and Fine Atmospheric Particulate Matter on a Mast Cell Line].

We investigated the cytotoxicity on a mast cell line (C57 cells) of water-soluble extracts of coarse (>3 µm, PM>3) and fine (0.05-3 µm, PM0.05-3) atmospheric particulates collected from April 2016 to March 2019 in Fukuoka, Japan. We examined the direct cytotoxicity with punched-out membrane filter fragments of PM>3 and PM0.05-3 collected from April 2019 to March 2021, without extraction of the components. Also, cell proliferation and degranulation assays were conducted under conditions which caused no cytotoxicity with water-soluble extracts of PM>3 from FY2016 and PM>3 direct samples from FY2019. The findings revealed the significant direct cytotoxicity of many PM>3 and all PM0.05-3 samples, with higher cytotoxicity for PM0.05-3 (FY2019-2020). These results were different from the cytotoxicity effects of water-soluble extracts of PM>3 and PM0.05-3 samples (FY2016) in previous studies. In addition, inhibition of cell proliferation and induction of degranulation were significantly induced in a few PM>3 samples, showing a correlation with the suspended particulate matter (SPM) concentrations. This method using punched-out membrane filters is convenient and useful for assessing the direct effects of atmospheric particles on a small scale.

Peptide-to-Small Molecule: Discovery of Non-Covalent, Active-Site Inhibitors of ß-Herpesvirus Proteases.

Viral proteases, the key enzymes that regulate viral replication and assembly, are promising targets for antiviral drug discovery. Herpesvirus proteases are enzymes with no crystallographically confirmed noncovalent active-site binders, owing to their shallow and polar substrate-binding pockets. Here, we applied our previously reported "Peptide-to-Small Molecule" strategy to generate novel inhibitors of ß-herpesvirus proteases. Rapid selection with a display technology was used to identify macrocyclic peptide 1 bound to the active site of human cytomegalovirus protease (HCMVPro) with high affinity, and pharmacophore queries were defined based on the results of subsequent intermolecular interaction analyses. Membrane-permeable small molecule 19, designed de novo according to this hypothesis, exhibited enzyme inhibitory activity (IC50 = 10-6 to 10-7 M) against ß-herpesvirus proteases, and the design concept was proved by X-ray cocrystal analysis.

Inositol phosphate kinase Vip1p interacts with histone chaperone Asf1p in Saccharomyces cerevisiae.

Histone eviction and deposition are critical steps in many nuclear processes. The histone H3/H4 chaperone Asf1p is highly conserved and is involved in DNA replication, DNA repair, and transcription. To identify the factors concerned with anti-silencing function 1 (ASF1), we purified Asf1p-associated factors from the yeast Saccharomyces cerevisiae by a GST pull-down experiment, and mass spectrometry analysis was performed. Several factors are specifically associated with Asf1p, including Vip1p. VIP1 is conserved from yeast to humans and encodes inositol hexakisphoshate and inositol heptakisphosphate kinase. Vip1p interacted with Asf1p as a dimer or in a complex with another protein(s). Deletion of VIP1 did not affect the interaction between Asf1p and other Asf1p-associated factors. An in vitro GST pull-down assay indicated a direct interaction between Asf1p and Vip1p, and the interaction between the two factors in vivo was detected by an immunoprecipitation experiment. Furthermore, genetic experiments revealed that VIP1 disruption increased sensitivity to 6-azauracil (6-AU), but not to DNA-damaging reagents in wild-type and ASF1-deleted strains. It is thought that 6-AU decreases nucleotide levels and reduces transcription elongation. These observations suggest that the association of Asf1p and Vip1p may be implicated in transcription elongation.

Effect of lipopolysaccharide on the xenobiotic-induced expression and activity of hepatic cytochrome P450 in mice.

Infection-associated inflammation can alter the expression levels and functions of cytochrome P450s (CYPs). Cyp gene expression is regulated by the activation of several nuclear receptors, including pregnane X receptor (PXR), constitutive androstane receptor (CAR), and aryl hydrocarbon receptor (AhR). These receptors can be activated by xenobiotics, including medicines. Here, to study the xenobiotic-induced fluctuations in CYP during inflammation, we examined the effect of lipopolysaccharide (LPS) treatment on the level of mRNAs encoding hepatic CYPs induced by xenobiotic-activated nuclear receptors, in mice. Both the mRNA induction of Cyp genes and the metabolic activities of CYP proteins were examined. LPS treatment caused a significant decrease in the induced expression of the mRNAs for Cyp3a11, 2c29, 2c55, and 1a2, but not for Cyp2b10. To assess the CYP enzymatic activities, CYP3A-mediated testosterone 6ß-hydroxylation and the intrinsic clearance (CL(int)) of nifedipine in liver microsomes were measured in mice treated with the xenobiotic pregnenolone-16alpha-carbonitrile (PCN) with or without LPS administration. Both assays revealed that the CYP3A activity, which was induced by PCN, declined significantly after LPS treatment, and this decline correlated with the Cyp3a11 mRNA level. In addition, we found that the mRNAs for interleukin (IL)-1ß and tumor necrosis factor (TNF) α were increased after treatment with LPS plus xenobiotics. Our findings demonstrated that LPS treatment reduces the PXR- and AhR-mediated, and possibly CAR-mediated Cyp gene expression and further suggest that these decreases are dependent on inflammatory cytokines in the liver.

Competence of an artificial bent DNA as a transcriptional activator in mouse ES cells.

Curved DNA structures with a left-handed superhelical conformation can activate eukaryotic transcription. However, their potency in transgene activation in embryonic stem (ES) cells has not been examined. T20 is an artificial curved DNA of 180 bp that serves as a transcriptional activator. We investigated the effect of T20 on transcription in mouse ES cell lines or hepatocytes differentiated from them. We established 10 sets of cell lines each harboring a single copy of the reporter construct. Each set comprised a T20-harboring cell line and a T20-less control cell line. Analyses showed that in ES cells and in hepatocytes originating from these cells, T20 both activated and repressed transcription in a manner that was dependent on the locus of reporter. The present and previous studies strongly suggest that in cells that have a strict gene regulation system, transcriptional activation by T20 occurs only in a transcriptionally active locus in the genome.

Macrocyclic Peptides as a Novel Class of NNMT Inhibitors: A SAR Study Aimed at Inhibitory Activity in the Cell.

Nicotinamide N-methyltransferase (NNMT), which catalyzes the methylation of nicotinamide, is a cytosolic enzyme that has attracted much attention as a therapeutic target for a variety of diseases. However, despite the considerable interest in this target, reports of NNMT inhibitors have still been limited to date. In this work, utilizing in vitro translated macrocyclic peptide libraries, we identified peptide 1 as a novel class of NNMT inhibitors. Further exploration based on the X-ray cocrystal structures of the peptides with NNMT provided a dramatic improvement in inhibitory activity (peptide 23: IC50 = 0.15 nM). Furthermore, by balance of the peptides' lipophilicity and biological activity, inhibitory activity against NNMT in cell-based assay was successfully achieved (peptide 26: cell-based IC50 = 770 nM). These findings illuminate the potential of cyclic peptides as a relatively new drug discovery modality even for intracellular targets.

Highly efficient chromatin transcription induced by superhelically curved DNA segments: the underlying mechanism revealed by a yeast system.

Superhelically curved DNA structures can strongly activate transcription in mammalian cells. However, the mechanism underlying the activation has not been clarified. We investigated this mechanism in yeast cells, using 108, 180, and 252 bp synthetic curved DNA segments. Even in the presence of nucleosomes, these DNAs activated transcription from a UAS-deleted CYC1 promoter that is silenced in the presence of nucleosomes. The fold-activations of transcription by these segments, relative to the transcription on the control that lacked such segments, were 51.4, 63.4, and 56.4, respectively. The superhelically curved DNA structures favored nucleosome formation. However, the translational positions of the nucleosomes were dynamic. The high mobility of the nucleosomes on the superhelically curved DNA structures seemed to influence the mobility of the nucleosomes formed on the promoter and eventually enhanced the access to the center region of one TATA sequence. Functioning as a dock for the histone core and allowing nucleosome sliding seem to be the mechanisms underlying the transcriptional activation by superhelically curved DNA structures in chromatin. The present study provides important clues for designing and constructing artificial chromatin modulators, as a tool for chromatin engineering.