A synthetic small molecule for rapid induction of multiple pluripotency genes in mouse embryonic fibroblasts.

Cellular reprogramming involves profound alterations in genome-wide gene expression that is precisely controlled by a hypothetical epigenetic code. Small molecules have been shown to artificially induce epigenetic modifications in a sequence independent manner. Recently, we showed that specific DNA binding hairpin pyrrole-imidazole polyamides (PIPs) could be conjugated with chromatin modifying histone deacetylase inhibitors like SAHA to epigenetically activate certain pluripotent genes in mouse fibroblasts. In our steadfast progress to improve the efficiency of SAHA-PIPs, we identified a novel compound termed, δ that could dramatically induce the endogenous expression of Oct-3/4 and Nanog. Genome-wide gene analysis suggests that in just 24 h and at nM concentration, δ induced multiple pluripotency-associated genes including Rex1 and Cdh1 by more than ten-fold. δ treated MEFs also rapidly overcame the rate-limiting step of epithelial transition in cellular reprogramming by switching "[Formula: see text]" the complex transcriptional gene network

Intracrine activity involving NAD-dependent circadian steroidogenic activity governs age-associated meibomian gland dysfunction

新たなイントラクライン機構を用いた加齢性眼疾患治療へ --眼局所のホルモンの加齢変化とサーカディアンリズムが鍵--. 京都大学プレスリリース. 2022-02-14.Canonically, hormones are produced in the endocrine organs and delivered to target tissues. However, for steroids, the concept of tissue intracrinology, whereby hormones are produced in the tissues where they exert their effect without release into circulation, has been proposed, but its role in physiology/disease remains unclear. The meibomian glands in the eyelids produce oil to prevent tear evaporation, which reduces with aging. Here, we demonstrate that (re)activation of local intracrine activity through nicotinamide adenine dinucleotide (NAD+)-dependent circadian 3β-hydroxyl-steroid dehydrogenase (3β-HSD) activity ameliorates age-associated meibomian gland dysfunction and accompanying evaporative dry eye disease. Genetic ablation of 3β-HSD nullified local steroidogenesis and led to atrophy of the meibomian gland. Conversely, reactivation of 3β-HSD activity by boosting its coenzyme NAD+ availability improved glandular cell proliferation and alleviated the dry eye disease phenotype. Both women and men express 3β-HSD in the meibomian gland. Enhancing local steroidogenesis may help combat age-associated meibomian gland dysfunction

Upregulation of ANGPTL6 in mouse keratinocytes enhances susceptibility to psoriasis

Psoriasis is a chronic inflammatory skin disease marked by aberrant tissue repair. Mutant mice modeling psoriasis skin characteristics have provided useful information relevant to molecular mechanisms and could serve to evaluate therapeutic strategies. Here, we found that epidermal ANGPTL6 expression was markedly induced during tissue repair in mice. Analysis of mice overexpressing ANGPTL6 in keratinocytes (K14-Angptl6 Tg mice) revealed that epidermal ANGPTL6 activity promotes aberrant epidermal barrier function due to hyperproliferation of prematurely differentiated keratinocytes. Moreover, skin tissues of K14-Angptl6 Tg mice showed aberrantly activated skin tissue inflammation seen in psoriasis. Levels of the proteins S100A9, recently proposed as therapeutic targets for psoriasis, also increased in skin tissue of K14-Angptl6 Tg mice, but psoriasis-like inflammatory phenotypes in those mice were not rescued by S100A9 deletion. This finding suggests that decreasing S100A9 levels may not ameliorate all cases of psoriasis and that diverse mechanisms underlie the condition. Finally, we observed enhanced levels of epidermal ANGPTL6 in tissue specimens from some psoriasis patients. We conclude that the K14-Angptl6 Tg mouse is useful to investigate psoriasis pathogenesis and for preclinical testing of new therapeutics. Our study also suggests that ANGPTL6 activation in keratinocytes enhances psoriasis susceptibility

Early Staphylococcal Biofilm Formation on Solid Orthopaedic Implant Materials: In Vitro Study

Biofilms forming on the surface of biomaterials can cause intractable implant-related infections. Bacterial adherence and early biofilm formation are influenced by the type of biomaterial used and the physical characteristics of implant surface. In this in vitro research, we evaluated the ability of Staphylococcus epidermidis, the main pathogen in implant-related infections, to form biofilms on the surface of the solid orthopaedic biomaterials, oxidized zirconium-niobium alloy, cobalt-chromium-molybdenum alloy (Co-Cr-Mo), titanium alloy (Ti-6Al-4V), commercially pure titanium (cp-Ti) and stainless steel. A bacterial suspension of Staphylococcus epidermidis strain RP62A (ATCC35984) was added to the surface of specimens and incubated. The stained biofilms were imaged with a digital optical microscope and the biofilm coverage rate (BCR) was calculated. The total amount of biofilm was determined with the crystal violet assay and the number of viable cells in the biofilm was counted using the plate count method. The BCR of all the biomaterials rose in proportion to culture duration. After culturing for 2-4 hours, the BCR was similar for all materials. However, after culturing for 6 hours, the BCR for Co-Cr-Mo alloy was significantly lower than for Ti-6Al-4V, cp-Ti and stainless steel (P0.05). These results suggest that surface properties, such as hydrophobicity or the low surface free energy of Co-Cr-Mo, may have some influence in inhibiting or delaying the two-dimensional expansion of biofilm on surfaces with a similar degree of smoothness

核酸の分子認識と光反応性に関する研究

京都大学0048新制・課程博士博士(理学)甲第17383号理博第3880号新制||理||1559(附属図書館)30149京都大学大学院理学研究科化学専攻(主査)教授 杉山 弘, 教授 三木 邦夫, 教授 藤井 紀子学位規則第4条第1項該当Doctor of ScienceKyoto UniversityDA

Sequence-specific DNA recognition by cyclic pyrrole-imidazole cysteine-derived polyamide dimers.

Pyrrole-imidazole (PI) polyamides bind to the minor groove of the DNA duplex in a sequence-specific manner and thus have the potential to regulate gene expression. To date, various types of PI polyamides have been designed as sequence-specific DNA binding ligands. One of these, cysteine cyclic PI polyamides containing two β-alanine molecules, were designed to recognize a 7 bp DNA sequence with high binding affinity. In this study, an efficient cyclization reaction between a cysteine and a chloroacetyl residue was used for dimerization in the synthesis of a unit that recognizes symmetrical DNA sequences. To evaluate specific DNA binding properties, dimeric PI polyamide binding was measured by using a surface plasmon resonance (SPR) method. Extending this molecular design, we synthesized a large dimeric PI polyamide that can recognize a 14 bp region in duplex DNA

Sequence-specific electron injection into DNA from an intermolecular electron donor.

Electron transfer in DNA has been intensively studied to elucidate its biological roles and for applications in bottom-up DNA nanotechnology. Recently, mechanisms of electron transfer to DNA have been investigated; however, most of the systems designed are intramolecular. Here, we synthesized pyrene-conjugated pyrrole-imidazole polyamides (PPIs) to achieve sequence-specific electron injection into DNA in an intermolecular fashion. Electron injection from PPIs into DNA was detected using 5-bromouracil as an electron acceptor. Twelve different 5-bromouracil-containing oligomers were synthesized to examine the electron-injection ability of PPI. Product analysis demonstrated that the electron transfer from PPIs was localized in a range of 8 bp from the binding site of the PPIs. These results demonstrate that PPIs can be a useful tool for sequence-specific electron injection

Photoreactivities of 5-Bromouracil-containing RNAs.

5-Bromouracil ((Br)U) was incorporated into three types of synthetic RNA and the products of the photoirradiated (Br)U-containing RNAs were investigated using HPLC and MS analysis. The photoirradiation of r(GCA(Br)UGC)(2) and r(CGAA(Br)UUGC)/r(GCAAUUCG) in A-form RNA produced the corresponding 2'-keto adenosine ((keto)A) product at the 5'-neighboring nucleotide, such as r(GC(keto)AUGC) and r(CGA(keto)AUUGC), respectively. The photoirradiation of r(CGCG(Br)UGCG)/r(C(m)GCAC(m)GCG) in Z-form RNA produced the 2'-keto guanosine ((keto)G) product r(CGC(keto)GUGCG), whereas almost no products were observed from the photoirradiation of r(CGCG(Br)UGCG)/r(C(m)GCAC(m)GCG) in A-form RNA. The present results indicate clearly that hydrogen (H) abstraction by the photochemically generated uracil-5-yl radical selectively occurs at the C2' position to provide a 2'-keto RNA product

Effect of single pyrrole replacement with β-alanine on DNA binding affinity and sequence specificity of hairpin pyrrole/imidazole polyamides targeting 5'-GCGC-3'.

N-Methylpyrrole (Py)-N-methylimidazole (Im) polyamides are small organic molecules that can recognize predetermined DNA sequences with high sequence specificity. As many eukaryotic promoter regions contain highly GC-rich sequences, it is valuable to synthesize and characterize Py-Im polyamides that recognize GC-rich motifs. In this study, we synthesized four hairpin Py-Im polyamides 1-4, which recognize 5'-GCGC-3' and investigated their binding behavior with surface plasmon resonance assay. Py-Im polyamides 2-4 contain two, one, and one β-alanine units, replacing the Py units of 1, respectively. The binding affinities of 2-4 to the target DNA increased 430, 390, and 610-fold, respectively, over that of 1. The association and dissociation rates of 2 to the target DNA were improved by 11 and 37-fold, respectively, compared with those of 1. Interestingly, the association and dissociation rates of 3 and 4 were higher than those of 2, even though the binding affinities of 2, 3, and 4 to the target DNA were comparable to each other. The binding affinity of 2 to DNA with a 2bp mismatch was reduced by 29-fold, compared with that to the matched DNA. Moreover, the binding affinities of 3 and 4 to the same mismatched DNA were reduced by 270 and 110-fold, respectively, indicating that 3 and 4 have greater specificities than 2 and are suitable as DNA-binding modules for engineered epigenetic regulation

Binding of hairpin pyrrole and imidazole polyamides to DNA: relationship between torsion angle and association rate constants.

N-methylpyrrole (Py)-N-methylimidazole (Im) polyamides are small organic molecules that bind to DNA with sequence specificity and can be used as synthetic DNA-binding ligands. In this study, five hairpin eight-ring Py-Im polyamides 1-5 with different number of Im rings were synthesized, and their binding behaviour was investigated with surface plasmon resonance assay. It was found that association rate (k(a)) of the Py-Im polyamides with their target DNA decreased with the number of Im in the Py-Im polyamides. The structures of four-ring Py-Im polyamides derived from density functional theory revealed that the dihedral angle of the Py amide carbonyl is 14∼18°, whereas that of the Im is significantly smaller. As the minor groove of DNA has a helical structure, planar Py-Im polyamides need to change their conformation to fit it upon binding to the minor groove. The data explain that an increase in planarity of Py-Im polyamide induced by the incorporation of Im reduces the association rate of Py-Im polyamides. This fundamental knowledge of the binding of Py-Im polyamides to DNA will facilitate the design of hairpin Py-Im polyamides as synthetic DNA-binding modules