Iridium-Catalyzed [2 + 2 + 2] Cycloaddition of Bithiophen-Linked Diynes with Nitriles: Scope and Mechanistic Study with Quantum Chemical Calculation.

We report a simple and atom-efficient method for the synthesis of bithiophene-fused isoquinolines by iridium-catalyzed [2 + 2 + 2] cycloaddition of bithiophene-linked diynes with nitriles. All three structural isomers of bithiophene-linked diynes underwent [2 + 2 + 2] cycloaddition, and the trend in the reactivity for cycloaddition was diyne 1 = diyne 3 > diyne 2. Dibenzothiophene-linked diyne also reacted with nitriles to form a variety of cycloadducts. Cycloaddition of bithiophene-linked diynes with alkynes and an isocyanate formed naphthodithiophenes and a 2-pyridone derivative, respectively. Cycloadducts bearing a 2-aminopyridine moiety and benzothiophene rings showed intense fluorescence at around 530 nm and gave a fluorescence quantum yield of 0.44. Furthermore, quantum chemical calculations provided insight into the origin of the difference in reactivity of three bithiophene-linked diynes. The different reactivities of the three diynes 1-3 are believed to originate from the step where an iridacyclopentadiene reacts with a coordinated nitrile to form azairidabicyclo[3.2.0]heptatriene. HOMOs of iridacyclopentadiene play a decisive role in this step.

Changes in the Expression of Smooth Muscle Cell-Related Genes in Human Dermal Sheath Cup Cells Associated with the Treatment Outcome of Autologous Cell-Based Therapy for Male and Female Pattern Hair Loss.

In a clinical study of autologous cell-based therapy using dermal sheath cup (DSC) cells, the treatment of hair loss showed improvements. However, the outcomes were variable. Here, correlations between marker gene expression in DSC cells and treatment outcomes were assessed to predict therapeutic efficacy. Overall, 32 DSC cell lines were used to evaluate correlations between marker gene expression and treatment outcomes. Correlations between vascular pericyte and preadipocyte marker expression and treatment outcomes were inconsistent. As smooth muscle cell markers, MYOCD correlated negatively with treatment outcomes and SRF consistently demonstrated an inverse correlation. Additionally, CALD1 correlated negatively and ACTA2 correlated inversely with treatment outcomes. DSC cell lines were divided into good and moderate/poor responders to further investigate the correlations. SRF and CALD1 were lower in a good responder compared with a moderate responder. Next, DSC cells were differentiated toward dermal papilla cells. Dermal papilla markers SOX2 and LEF1 before differentiation had moderate positive and inverse correlations with the treatment outcome, respectively. SOX2 after differentiation more consistently demonstrated a positive correlation. Significant downregulation of smooth muscle-related genes was also observed after differentiation. These findings revealed putative markers for preclinical evaluation of DSC cells to improve hair loss.

Characterization of human dermal sheath cells reveals CD36-expressing perivascular cells associated with capillary blood vessel formation in hair follicles.

Dermal sheath (DS) is located at the outermost border of hair follicles, comprising the connective tissue sheath of these follicles; DS cells are known to contribute to hair cycling and follicle neogenesis. However, the mechanisms by which DS cells contribute to hair formation are currently unclear. We investigated the global transcriptional profile of human DS cells in early passaged culture, compared with those of human dermal papilla cells (DP cells) and dermal fibroblasts. Vascular related genes were highly expressed in DS cells, and expression of the multi-ligand receptor, CD36, was significantly higher in DS cells than in DP cells. Further analyses with whole-mount imaging technique showed that dense networks of blood capillaries were formed in the DS of human anagen hair follicles, whereas regression of blood capillaries was observed in telogen and catagen hair follicles. We found that CD36-expressing cells were present in populations of DS cells, but were rarely observed in populations of DP cells and fibroblasts. Furthermore, our results indicated that CD36-expressing DS cells may participate in angiogenesis. Therefore, we concluded that CD36-expressing DS cells may modulate blood capillaries in hair follicles, in association with hair cycling.

Wnt activator CHIR99021-stimulated human dermal papilla spheroids contribute to hair follicle formation and production of reconstituted follicle-enriched human skin.

The aim of the present study was to accomplish de novo generation of reconstituted human skin with enriched hair follicles. Dermal papillae (DP) are known to play a crucial organizing role in hair follicle induction. However, generation of enriched human hair follicles using cultured DP cells has not been accomplished because DP cells easily lose their hair-inducing ability with culturing. To enhance the hair-inducing ability of DP cells, Wnt signaling pathway activation or three-dimensional (3D) spheroid culture methods were employed in previous studies. Herein, we assessed effects of the canonical Wnt/ß-catenin signaling activator CHIR99021 and found that it enhanced the expression of DP signature genes associated with hair-inducing ability. Further comparison of three different 3D culture methods revealed the highest expression of DP signature genes in spheroids generated by a floating drop method compared with other methods. CHIR99021 synergistically increased expression of DP signature genes in combination with floating drop culture. "Reconstituted skin assay" prepared using the most promising CHIR99021-stimulated 3D spheroids showed enrichment for human hair follicles. Labeled DP spheroids and derived cells were primarily found to be DP and dermal sheath cup (DSC) cells, implying organization of hair formation by DP spheroids. Finally, to evaluate the functional features of generated human skin and hair follicles, we injected human DSC cells, which reportedly show DP precursor behavior, and exhibit hair-inducing ability through incorporation into hair follicles, into mice. Histological studies revealed injected DSC cells in dermal sheath of hair follicles, consistent with a previous report, thus verifying the functionality of generated skin and hair follicles. Collectively, our findings demonstrate that DP spheroids synergistically stimulated by CHIR99021 and 3D culture contributed to hair follicle formation, thus making it possible to generate reconstituted hair follicle-enriched human skin with functional features.

Synthesis of Multisubstituted Azatriphenylenes by Iridium-Catalyzed [2 + 2 + 2] Cycloaddition of Biaryl-Linked Diynes with Nitriles.

A convenient synthesis of multisubstituted azatriphenylenes is reported. [Ir(cod)Cl]2/diphosphine is an efficient catalyst for the [2 + 2 + 2] cycloaddition of biaryl-linked diynes with nitriles to give multisubstituted azatriphenylenes in high yields. Aromatic, heteroaromatic, aliphatic, and functionalized nitriles could be used for the reaction.

Gene Expression Profiling of the Intact Dermal Sheath Cup of Human Hair Follicles.

Cells that constitute the dermal papillae of hair follicles might be derived from the dermal sheath, the peribulbar component of which is the dermal sheath cup. The dermal sheath cup is thought to include the progenitor cells of the dermal papillae and possesses hair inductive potential; however, it has not yet been well characterized. This study investigated the gene expression profile of the intact dermal sheath cup, and identified dermal sheath cup signature genes, including extracellular matrix components and bone morphogenetic protein-binding molecules, as well as transforming frowth factor beta 1 as an upstream regulator. Among these, gremilin-2, a member of the bone morphogenetic protein antagonists, was found by in situ hybridization to be highly specific to the dermal sheath cup, implying that gremlin-2 is a key molecule contributing to maintenance of the properties of the dermal sheath cup.

The Pou5f1/Pou3f-dependent but SoxB-independent regulation of conserved enhancer N2 initiates Sox2 expression during epiblast to neural plate stages in vertebrates.

The transcription factor Sox2 is a core component of the pluripotency control circuits in the early embryo, and later controls many aspects of neural development. Here, we demonstrate that Sox2 expression in the epiblast (mouse blastoderm) and anterior neural plate (ANP) is determined by the upstream enhancer N2. The mouse enhancer N2 exhibits strong activity in mouse ES cells, epiblast and ANP, and is regulated correctly in chicken and zebrafish embryos. Targeted deletion of this enhancer in mouse embryos caused a large reduction of Sox2 expression to 10% of that of wild-type levels in epiblast and ANP. However, this was tolerated by mouse embryo, probably due to functional compensation by Sox3. The activity of enhancer N2 depends on phylogenetically conserved bipartite POU factor-binding motifs in a 73-bp core sequence that function synergistically, but this activation does not involve Sox2. The major POU factor expressed at the epiblastic stage is Pou5f1 (Oct3/4), while those in the anterior neural plate are Pou3f factors (Oct6, Brn2 etc.). These factors are gradually exchanged during the transition from epiblast to ANP stages in mouse embryos and epiblast stem cells (EpiSC). Consistently, enhancer N2 activity changes from full Pou5f1 dependence to Pou3f dependence during the development of neural plate cells (NPC) from EpiSC, as assessed by specific POU factor knockdown in these cells. Zebrafish mutant embryos completely devoid of Pou5f1 activity failed to activate enhancer N2 and to express Sox2 in the blastoderm and ANP, and these defects were rescued by exogenous supply of pou5f1. Previously, Pou5f1-Sox2 synergism-dependent Sox2 activation through enhancer SRR2 in ES cells has been highlighted, but this mechanism is limited to ES cells and amniotes. In contrast, the enhancer N2-mediated, POU factor-dependent activation of Sox2, without involvement of Sox2, is a phylogenetically conserved core mechanism that functions in gene regulatory networks at early embryonic stages.

ß-Amyrin oxidation by oat CYP51H10 expressed heterologously in yeast cells: the first example of CYP51-dependent metabolism other than the 14-demethylation of sterol precursors.

CYP51 has been recognized as a unique CYP family that consists of one isolated molecular species, a sterol 14-demethylase essential for sterol biosynthesis. However, another CYP51 gene classified as the CYP51H subfamily has been identified in higher plants, in addition to a sterol 14-demethylase gene, CYP51G1. To shed light on the function of this "second CYP51", oat CYP51H10 was introduced into the ß-amyrin-producing yeast cells, and the effect of the expressed CYP51H10 on ß-amyrin metabolism in the host cells was examined. In the CYP51H10-introduced cells, ß-amyrin was converted to a metabolite with 12,13-epoxy and one additional hydroxyl group. Since the 12,13-epoxy group introduced into ß-amyrin ring is an essential structure of avenacin A-1, a triterpene glycoside produced in oat from ß-amyrin, the present findings indicate the contribution of CYP51H10 to avenacin A-1 biosynthesis from ß-amyrin. This is the first study showing a second function of the CYP51 family.

Characterization of moss ent-kaurene oxidase (CYP701B1) using a highly purified preparation.

CYP701B1 of the moss, Physcomitrella patents, might be a unique cytochrome P450 having the ent-kaurene oxidase (KO) activity occurring in nonvascular plant. Phylogenetic analysis suggested that the gene encoding CYP701B1 was diverged from a common ancestral gene encoding KO of vascular plants. CYP701B1 expressed in Phichia yeast microsomes was purified and characterized. The purified CYP701B1 catalyzed the oxidation of ent-kaurene to ent-kaurenoic acid through three successive monooxygenations, and the rate-limiting step of this oxidation might be the initial step that forms ent-kaurenol. CYP701B1 was a typical ferric low-spin cytochrome P450 and was completely moved to high-spin state upon binding with ent-kaurene, and apparent Kd of ent-kaurene estimated by the spectral change caused by this spin-state shift was 2.5 µM. The potent KO inhibitor uniconazole, an azole compound with molecular size similar to ent-kaurene, bound CYP701B1 with high affinity. However, ketoconazole, an azole compound whose molecular size is larger than ent-kaurene could not bind to CYP701B, though it binds strongly with CYP51, lanosterol 14-demethylase. The results indicated that the active site of CYP701B1 is fitted for the molecular size of ent-kaurene. The P450 monooxygenase adapted for ent-kaurene oxidation might appear in land plants before evolutionary divergence into vascular and nonvascular plants.

Oxidative metabolism of 5-methoxy-N,N-diisopropyltryptamine (Foxy) by human liver microsomes and recombinant cytochrome P450 enzymes.

In vitro quantitative studies of the oxidative metabolism of (5-methoxy-N,N-diisopropyltryptamine, 5-MeO-DIPT, Foxy) were performed using human liver microsomal fractions and recombinant CYP enzymes and synthetic 5-MeO-DIPT metabolites. 5-MeO-DIPT was mainly oxidized to O-demethylated (5-OH-DIPT) and N-deisopropylated (5-MeO-IPT) metabolites in pooled human liver microsomes. In kinetic studies, 5-MeO-DIPT O-demethylation showed monophasic kinetics, whereas its N-deisopropylation showed triphasic kinetics. Among six recombinant CYP enzymes (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) expressed in yeast or insect cells, only CYP2D6 exhibited 5-MeO-DIPT O-demethylase activity, while CYP1A2, CYP2C8, CYP2C9, CYP2C19 and CYP3A4 showed 5-MeO-DIPT N-deisopropylase activities. The apparent Km value of CYP2D6 was close to that for 5-MeO-DIPT O-demethylation, and the Km values of other CYP enzymes were similar to those of the low-Km (CYP2C19), intermediate-Km (CYP1A2, CYP2C8 and CYP3A4) and high-Km phases (CYP2C9), respectively, for N-deisopropylation in human liver microsomes. In inhibition studies, quinidine (1 microM), an inhibitor of CYP2D6, almost completely inhibited human liver microsomal 5-MeO-DIPT O-demethylation at a substrate concentration of 10 microM. Furafylline, a CYP1A2 inhibitor, quercetin, a CYP2C8 inhibitor, sulfaphenazole, a CYP2C9 inhibitor and ketoconazole, a CYP3A4 inihibitor (5 microM each) suppressed about 60%, 45%, 15% and 40%, respectively, of 5-MeO-DIPT N-deisopropylation at 50 microM substrate. In contrast, omeprazole (10 microM), a CYP2C19 inhibitor, suppressed only 10% of N-deisopropylation by human liver microsomes, whereas at the same concentration the inhibitor suppressed the reaction by recombinant CYP2C19 almost completely. These results indicate that CYP2D6 is the major 5-MeO-DIPT O-demethylase, and CYP1A2, CYP2C8 and CYP3A4 are the major 5-MeO-DIPT N-deisopropylase enzymes in the human liver.

Effects of Y132H and F145L substitutions on the activity, azole resistance and spectral properties of Candida albicans sterol 14-demethylase P450 (CYP51): a live example showing the selection of altered P450 through interaction with environmental compounds.

Three variants of Candida albicans CYP51 (sterol 14-demethylase P450) having Y132H and/or F145L substitutions were purified and characterized to reveal the effects of these amino acid substitutions on the enzymatic properties and azole resistance of the enzyme. Y132H and F145L substitutions modified the spectral properties of the enzyme, suggesting that they caused some structural change modifying the heme environments of CYP51. Y132H and F145L substitutions increased the resistance of the enzyme to azole compounds but considerably decreased the catalytic activity. This fact represents a trade-off between acquisition of azole resistance and maintenance of high activity in the CYP51 having Y132H and F145L substitutions. A fluconazole-resistant C. albicans strain DUMC136 isolated from patients receiving long-term azole treatment was a homozygote of the altered CYP51 having Y132H and F145L substitutions. However, neither of these substitutions was found in CYP51 of wild-type C. albicans so far studied. These facts suggest that the azole-resistant variant having Y132H and/or F145L substitutions might be selected only under azole-rich environments because of its azole resistance and impaired catalytic activity. This may be a live example showing one of the important processes of P450 diversification, the selection of altered P450 through the interaction with environmental compounds.

Structure-based de novo design, synthesis, and biological evaluation of non-azole inhibitors specific for lanosterol 14alpha-demethylase of fungi.

The active site of lanosterol 14alpha-demethylase (CYP51) was investigated via MCSS functional group mapping and LUDI calculations. Several non-azole lead molecules were obtained by coupling structure-based de novo design with chemical synthesis and biological evaluation. All of the lead molecules exhibited a strong inhibitory effect on CYP51 of Candida albicans. They occupy the substrate-binding site and interfere with the binding of azole antifungal agents in a competitive manner. The mode of action of the lead molecules was validated by spectrophotomeric analysis and SAR studies. This is the first successful example reported for the inhibitor design of the cytochrome P450 superfamily using the de novo design strategy. Because the affinity of the lead molecules for CYP51 was mainly attributed to their nonbonding interaction with the apoprotein, the studies presented here afford the opportunity to develop novel antifungal agents that specifically interact with the residues in the active site and avoid the serious toxicity arising from coordination binding with the heme of mammalian P450s.

Urokinase-type plasminogen activator is activated in stratum corneum after barrier disruption.

The plasminogen/plasmin system in epidermis is thought to be the major protease involved in the delay of barrier recovery. However, little is known about the mechanism through which this system is activated. In order to clarify this mechanism, we first determined the distribution of proteolytic activity by using in situ zymography. As a result, plasminogen-activator activity was found to be present in the stratum corneum (SC) after barrier disruption. Next, SC subjected to repeated barrier disruption was collected to identify the protease. The protease was identified as urokinase-type plasminogen activator, because flybrinolytic activity of the collected SC was abolished by addition of anti-urokinase antibody. Urokinase activation in SC was confirmed by means of an in vitro assay, in which the precursor of urokinase (pro-uPA) became active after incubation with the insoluble component of SC homogenate. These findings indicated that urokinase-type plasminogen activator is activated in SC after barrier disruption and this activation might trigger the plasminogen/plasmin system in the epidermis.

Studies on the expression levels of sterol-metabolizing enzymes in the obese model SHR/NDmcr-cp rats.

1. Expression levels of four key enzymes of cholesterol metabolism, namely 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, lanosterol 14-demethylase (CYP51), cholesterol 7alpha-hydroxylase (CYP7A1) and sterol 12alpha-hydroxylase (CYP8B1), in metabolic syndrome model rats (SHR/NDmcr-cp) were examined. 2. Decreased expression of CYP51, which may be linked to the development of obesity, was found in the rats. 3. Expression of CYP8B1 was significantly higher in young rats. 4. No substantial change was observed in the mRNA levels of the dominant rate-limiting enzymes of sterol metabolism, namely HMG-CoA reductase and CYP7A1, in the rats. 5. These findings suggest that the expression levels of two key enzymes managing the downstream parts of the cholesterol-metabolizing pathways are altered in the rats, although little change was observed in the expression levels of the dominant rate-limiting enzymes of cholesterol metabolism.

Localization of serine racemase and its role in the skin.

D-serine is an endogenous coagonist of the N-methyl-D-aspartate (NMDA)-type glutamate receptor in the central nervous system and its synthesis is catalyzed by serine racemase (SR). Recently, the NMDA receptor has been found to be expressed in keratinocytes (KCs) of the skin and involved in the regulation of KC growth and differentiation. However, the localization and role of SR in the skin remain unknown. Here, using SR-knockout (SR-KO) mice as the control, we demonstrated the localization of the SR protein in the granular and cornified layer of the epidermis of wild-type (WT) mice and its appearance in confluent WT KCs. We also demonstrated the existence of a mechanism for conversion of L-serine to D-serine in epidermal KCs. Furthermore, we found increased expression levels of genes involved in the differentiation of epidermal KCs in adult SR-KO mice, and alterations in the barrier function and ultrastructure of the epidermis in postnatal day 5 SR-KO mice. Our findings suggest that SR in the skin epidermis is involved in the differentiation of epidermal KCs and the formation of the skin barrier.