Various physiological and pathological processes are accompanied with the alteration of pH at extracellular juxtamembrane region. Accordingly, the methods to analyze the cell surface pH have been demanded in biological and medical sciences. In this study, we have established a novel methodology for cell surface pH imaging using poly(ethylene glycol)-phospholipid (PEG-lipid) as a core structure of ratiometric fluorescent probes. PEG-lipid is a synthetic amphiphilic polymer originally developed for the cell surface modification in transplantation therapy. Via its hydrophobic alkyl chains of the phospholipid moiety, PEG-lipid is, when applied extracellularly, spontaneously inserted into the plasma membrane and retained at the surface of the cells. We have demonstrated that the PEG-lipid conjugated with fluorescein isothiocyanate (FITC-PEG-lipid) can be used as a sensitive and reversible cell-surface-anchored pH probe between weakly alkaline and acidic pH with an excellent spatiotemporal resolution. The remarkably simple procedure for cell-surface labeling with FITC-PEG-lipid would also be advantageous when considering its application to high-throughput in vitro assay. This study further indicates that various probes useful for the investigation of juxtamembrane environments could also be developed by using PEG-lipid as the core structure for bio-membrane anchoring.

Endochondral ossification, including bone growth and other metabolic events, is regulated by circadian rhythms. Herein, we provide evidence that melatonin has a direct effect on the circadian rhythm of chondrocytes. We detected mRNA expression of the genes which encode the melatonin-synthesizing enzymes AANAT (arylalkylamine N-acetyltransferase) and HIOMT (hydroxyindole O-methyltransferase), as well as the melatonin receptors MT1 and MT2 in mouse primary chondrocytes and cartilage. Production of melatonin was confirmed by mass spectrometric analysis of primary rat and chick chondrocytes. Addition of melatonin to primary mouse chondrocytes caused enhanced cell growth and increased expression of Col2a1, Aggrecan, and Sox9, but inhibited Col10a1 expression in primary BALB/c mouse chondrocytes. Addition of luzindole, an MT1 and MT2 antagonist, abolished these effects. These data indicate that chondrocytes produce melatonin, which regulates cartilage growth and maturation via the MT1 and MT2 receptors. Kinetic analysis showed that melatonin caused rapid upregulation of Aanat, Mt1, Mt2, and Pthrp expression, followed by Sox9 and Ihh. Furthermore, expression of the clock gene Bmal1 was induced, while that of Per1 was downregulated. Chronobiological analysis of synchronized C3H mouse chondrocytes revealed that melatonin induced the cyclic expression of Aanat and modified the cyclic rhythm of Bmal1, Mt1, and Mt2. In contrast, Mt1 and Mt2 showed different rhythms from Bmal1 and Aanat, indicating the existence of different regulatory genes.Our results indicate that exogenous and endogenous melatonin work in synergy in chondrocytes to adjust rhythmic expression to the central suprachiasmatic nucleus clock.

This article reports a new route to access spirocyclic 2‐oxindoles containing the α‐methylene‐γ‐butyrolactam structure via “electrophilic amide allylation”. The key reaction was accomplished by using acetoxy methacrylamides and tetrakis(triphenylphosphine)palladium as catalyst, affording a variety of the amide allylated products in excellent yields. The successful cyclization of these products has demonstrated the potential utility of this approach to offer a practical synthesis of spirocyclic oxindoles.

The oxetane and the non-oxetane mechanisms of the electron-transfer-driven repair of the (64) TT photolesion of DNA by photolyase are examined by density functional theory (B3LYP). We calculated the radical cation pathway in addition to the radical anion and the neutral pathways for both mechanisms in order to assess the possibility of the radical cation pathway, because relatively large energy barriers have been found for the radical anion pathway. As a result, the radical anion pathway showed a large energy barrier in both the oxetane and the non-oxetane mechanisms in agreement with previous calculations. However, it was found that the radical cation pathway of the oxetane mechanism has a realistic low energy barrier. This advantage of the radical cation pathway was ascribed to the position of the radical before the formation of the oxetane and the stability of the oxetane in energy.DNA is highly a reactive substance and is therefore readily influenced and damaged, because the inside of the cell is under chemically active conditions due to ultraviolet rays, chemical substances, and so on. In fact, DNA is routinely damaged and various types of DNA lesions are known.1 Dimerized adjacent thymines, classified as single-strand damage, is also one of such DNA lesions. Although DNA lesions can be an origin of diseases such as cancer, damaged DNA is immediately recovered through a repair process 1 in order to maintain normal genetic information.For example, formed thymine dimer is repaired by photolyase under photoirradiation in plants and bacteria.2 Many people have focused on this repair process as a model and have endeavored to understand its mechanism.3 There exist two forms of thymine dimer, cyclobutane pyrimidine dimer (CPD) and (64) photoproduct (Figure 1), and many examinations of the repair mechanism have been conducted especially for the case of CPD. Photolyase is a flavoprotein that has a chromophore cofactor flavin adenine dinucleotide (FAD) (Figure 1) playing an important role as a coenzyme. A second chromophore cofactor that functions as an antenna harvesting blue light, methenyltetrahydrofolate (MTHF) or , is also present inside the protein. The FAD, which is thought to be fully reduced to FADH ¹ inside the protein, is buried in the vicinity of the active site. On the other hand, the second cofactor is further deeply buried and a little far from FAD.The generally proposed mechanism of the repair reaction of the thymine dimer on the basis of previous findings for CPD is displayed in Figure 2A. The function of the photolyase originates in the blue-light harvest by the second chromophore cofactor. The electronic state of the second cofactor that absorbed the light is enhanced to an excited state and this excitation energy is transmitted to the other cofactor FADH

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