Direct observation of photoinduced sequential spin transition in a halogen-bonded hybrid system by complementary ultrafast optical and electron probes.

A detailed understanding of the ultrafast dynamics of halogen-bonded materials is desired for designing supramolecular materials and tuning various electronic properties by external stimuli. Here, a prototypical halogen-bonded multifunctional material containing spin crossover (SCO) cations and paramagnetic radical anions is studied as a model system of photo-switchable SCO hybrid systems using ultrafast electron diffraction and two complementary optical spectroscopic techniques. Our results reveal a sequential dynamics from SCO to radical dimer softening, uncovering a key transient intermediate state. In combination with quantum chemistry calculations, we demonstrate the presence of halogen bonds in the low- and high-temperature phases and propose their role during the photoinduced sequential dynamics, underscoring the significance of exploring ultrafast dynamics. Our research highlights the promising utility of halogen bonds in finely tuning functional properties across diverse photoactive multifunctional materials.

[Aging-associated Cyst Formation and Fibrosis].

Cysts are abnormal fluid-filled sacs found in various human organs, including the liver. Liver cysts can be associated with known causes such as parasite infections and gene mutations, or simply aging. Among these causes, simple liver cysts are often found in elderly people. While they are generally benign, they may occasionally grow but rarely shrink with age, indicating their clear association with aging. However, the mechanism behind the formation of simple liver cysts has not been thoroughly investigated. Recently, we have generated transgenic mice that specifically overexpress fibroblast growth factor (FGF)18 in hepatocytes. These mice exhibit severe liver fibrosis without inflammation and spontaneously develop liver cysts that grow with age. Our findings suggest that simple liver cysts can be induced by fibrosis accompanied by sterile inflammation or injury, whereas fibrosis accompanied by severe inflammation or injury may lead to cirrhosis. We also discuss the detrimental effects of disease- and aging-associated fibrosis in various organs, such as the heart, lungs, and kidneys. Additionally, we provide a brief summary of the two currently approved anti-fibrotic drugs for idiopathic pulmonary fibrosis, nintedanib and pirfenidone, as well as their possibility of future expansion of application toward other fibrotic diseases.

[Dissemination of Information from Basic Research on Aging: Species, Strain, Substrain and Sex Differences].

Various organisms with different lifespans such as yeast, nematodes, fruit fly, mice, and rats are used for basic research on mechanisms of aging and anti-aging. These organisms are often genetically engineered and used to elucidate the contribution of certain genes to aging. For example, genetic recombination techniques revealed that the lifespan of superoxide dismutase (SOD) transgenic flies extended up to approximately 30%. This result suggests that increasing antioxidant capacity extends lifespan possibly by reducing oxidative damage. However, a similar experiment conducted in mice did not shown any positive effect of prolonging lifespan. Likewise, differences between animal species have also been observed in administration experiments of antioxidants such as resveratrol and curcumin. Further complicating the understanding of aging processes are differences among substrain and sex differences. For instance, the activity of catalase (CAT) in rat liver decreases with age in males, but increases in females. In this review, we describe the diversity of age-related changes, focusing on species, strain/substrain, and sex differences and introduce some efforts to address these issues in aging research.

Direct Fluorescence Evaluation of d-Amino Acid Oxidase Activity Using a Synthetic d-Kynurenine Derivative.

d-Amino acid oxidase (DAO) has been suggested to be associated with the central nervous system diseases, such as schizophrenia. We newly synthesized a nonfluorescent 5-methylthio-d-kynurenine (MeS-d-KYN), which was converted to blue-fluorescent 6-MeS-kynurenic acid (MeS-KYNA, λex = 364 nm, λem = 450 nm) through a one-step reaction by incubation with DAO. It was revealed that fluorescence intensity increased accompanied by commercial porcine kidney DAO activity (unit) with a good correlation (R2 = 0.9972), suggesting that the fluorometric evaluation of DAO activity using MeS-d-KYN is feasible. MeS-d-KYN was applied to fluorescent DAO imaging in cultured LLC-PK1 cells, and the blue fluorescence of MeS-KYNA overlapped considerably with the location of peroxisomes, which was suggested to be the location of DAO in the cells. Because fluorescence was diminished in the presence of 6-chloro-1,2-benzisoxazol-3(2H)-one (CBIO), a DAO inhibitor, it was considered that DAO activity in cells could be directly evaluated using MeS-d-KYN as the substrate.

[Multidisciplinary Approach to Aging].

Age-related decreases of various physiological functions have significant influence on activities of daily living (ADL) and QOL in elderly populations. Mechanisms of aging are currently the focus of many researchers in a wide range of studies. Researchers are trying to find novel ways to attenuate or delay aging in humans as well as to develop interventions for age-associated diseases. In this review, we briefly discuss the need for a multidisciplinary approach in aging research.

Synthesis, characterization, and supramolecular architectures of two distinct classes of probes for the visualization of endogenously generated hypochlorite ions in response to cellular activity.

Two distinct classes of compounds, (E)-2-(((3-amino-4-nitrophenyl) imino) methyl)-5-(diethylamino) phenol (SB) and 5-(diethylamino)-2-(5-nitro-1H-benzo[d]imidazol-2-yl) phenol (IM) were synthesized. SB, a bright red colored compound was crystallized in acetonitrile as a triclinic crystal system while IM, yellow colored compound crystallized as a monoclinic crystal system in dimethylformamide by vapor diffusion of diethylether. These compounds were characterized using spectroscopic techniques (IR, UV-visible, 1H, and 13C NMR), and X-ray crystallography. SB and IM displayed classical and non-classical H-bonding involving C-H…O and π…π interactions. These compounds detected hypochlorite ions in aqueous DMSO (1: 9, v/v, HEPES buffer, pH 7.4), and detection was visible via color changes by naked eye. We also performed UV-visible and fluorescence titrations, showing detection limits of 8.82 × 10-7 M for SB and 2.44 × 10-7 M for IM. The fluorometric responses from SB and IM were also studied against different ROS and anions. DFT calculations were performed to strengthen the proposed sensing mechanisms of both SB and IM. Hypochlorite, which is endogenously generated by myeloperoxidase in endosomes, was specifically visualized using SB and IM in lipopolysaccharide-treated RAW264.7 cells. These probes were also used to image the generation of hypochlorite by RAW264.7 cells during phagocytosis of non-fluorescent polystyrene beads.

Functional characterization of the neuron-restrictive silencer element in the human tryptophan hydroxylase 2 gene expression.

Tryptophan hydroxylase 2 (TPH2) is the key enzyme in the synthesis of neuronal serotonin. Although previous studies suggest that TPH2 neuron-restrictive silencer element (NRSE) functions as a negative regulator dependent on neuron-restrictive silencer factor (NRSF) activity, the underlying mechanisms are yet to be fully elucidated. Here, we show a detailed analysis of the NRSE-mediated repression of the human TPH2 (hTPH2) promoter activity in RN46A cells, a cell line derived from rat raphe neurons. Quantitative real-time RT-PCR analysis revealed the expression of serotonergic marker genes (Mash1, Nkx2.2, Gata2, Gata3, Lmx1b, Pet-1, 5-Htt, and Vmat2) and Nrsf gene in RN46A cells. Tph1 mRNA is the prevalent form expressed in RN46A cells; Tph2 mRNA is also expressed but at a lower level. Electrophoretic mobility shift assays and reporter assays showed that hTPH2 NRSE is necessary for the efficient DNA binding of NRSF and for the NRSF-dependent repression of the hTPH2 promoter activity. The hTPH2 promoter activity was increased by knockdown of NRSF, or over-expression of the engineered NRSF (a dominant-negative mutant or a DNA-binding domain and activation domain fusion protein). MS-275, a class I histone deacetylase (HDAC) inhibitor, was found to be more potent than MC-1568, a class II HDAC inhibitor, in enhancing the hTPH2 promoter activity. Furthermore, treatment with the ubiquitin-specific protease 7 deubiquitinase inhibitors, P-22077 or HBX 41108, increased the hTPH2 promoter activity. Collectively, our data demonstrate that the hTPH2 NRSE-mediated promoter repression via NRSF involves class I HDACs and is modulated by the ubiquitin-specific protease 7-mediated deubiquitination and stabilization of NRSF.

SAMP8 mice as a neuropathological model of accelerated brain aging and dementia: Toshio Takeda's legacy and future directions.

Senescence accelerated mice P8 (SAMP8) show significant age-related deteriorations in memory and learning ability in accordance with early onset and rapid advancement of senescence. Brains of SAMP8 mice reveal an age-associated increase of PAS-positive granular structures in the hippocampal formation and astrogliosis in the brain stem and hippocampus. A spongy degeneration in the brain stem appears at 1 month of age and reaches a maximum at 4-8 months. In addition, clusters of activated microglia also appear around the vacuoles in the brain stem. ß/A4(Aß) protein-like immunoreactive granular structures are observed in various regions and increase in number markedly with age. Other age-associated histological changes include cortical atrophy, neuronal cell loss in locus coeruleus and lateral tegmental nuclei, intraneuronal accumulation of lipopigments in Purkinje cells and eosinophilic inclusion bodies in thalamic neurons. A blood-brain barrier dysfunction and astrogliosis are also prominent with advancing age in the hippocampus. These changes are generally similar to the pathomorphology of aging human brains and characterized by their association with some specific glioneuronal reactions. As for the hallmarks of Alzheimer brains, tau morphology has not yet been confirmed regardless of the age-related increase in phosphorylated tau in SAMP8 mice brains, but early age-related Aß deposition in the hippocampus has recently been published. SAMP8 mice are, therefore, not only a senescence-accelerated model but also a promising model for Alzheimer's disease and other cognitive disorders.

Single-Molecule Fluorescence Spectroscopy of Perylene Diimide Dyes in a γ-Cyclodextrin Film: Manifestation of Photoinduced H-Atom Transfer via Higher Triplet (n, π*) Excited States.

Supramolecular complexation of γ-cyclodextrin (γ-CD) with N,N'-bis(2,6-dimethylphenyl)perylene-3,4,9,10-tetracarboxylic diimide (DMP-PDI) or N,N'-bis(2,6-dioctyl)perylene-3,4,9,10-tetracarboxylic diimide (C8-PDI) dye in an aqueous solution and in a γ-CD solid film were investigated via ensemble and single-molecule fluorescence spectroscopy. These two perylene diimide derivatives possess almost the same electronic structure but have different terminal functional groups. This structural difference leads to formation of an inclusion complex of γ-CD with DMP-PDI but not with C8-PDI in aqueous solution. In a γ-CD solid film, the distributions of the wavelengths of emission maximum (λmaxem) are strikingly different between these two dyes; a much narrower and blue-shifted λmaxem distribution was observed for C8-PDI relative to DMP-PDI. This difference is attributed to the fact that the C8-PDI molecules are bound at the γ-CD/glass interface as a result of spin-coating of the sample solution, whereas the DMP-PDI molecules form 1:1 and 1:2 inclusion complexes with conformational heterogeneities in the film. In comparison to the case for C8-PDI, more frequent on-off blinking events were observed for DMP-PDI. The blinking statistics of DMP-PDI in the γ-CD film exhibit both single-exponential and nonexponential (i.e., dispersive) kinetics, revealed by robust statistical analysis. Energetic consideration with the aid of theoretical calculations suggests that the underlying photophysics most probably involves hydrogen atom transfer (HAT) between the DMP-PDI guest and γ-CD host via higher excited (n, π*) triplet states. The hypothesis of HAT in the inclusion complex reasonably explains the experimental results; however, a charge transfer hypothesis cannot explain the results. The dispersive kinetics is attributable to the effect of thermal fluctuation in the forward and backward HAT reactions.

[An overview of current research of the effect of foods on aging and stress].

  The aging process is largely influenced by dietary factors. For example, caloric restriction can slow age-related functional deterioration and the onset or progression of age-related diseases, as well as prolong mean and maximum life span in laboratory animals. However, the dietary factors that affect the aging process comprise not only calories, but also various nutrients, such as proteins, carbohydrates, fats, and vitamins. Phytochemicals, which are found in plants, are non-nutritive, yet many phytochemicals are known to act as antioxidants and prevent diseases associated with free radical production. Furthermore, certain phytochemicals can help prevent or reduce the risk of cancer, inflammation, and cardiovascular disease by alteration of several signal transduction pathways in cells. Therefore, much focus is being placed on the effects of dietary phytochemicals on aging and stress response. This paper reviews recent advances in the study of two major dietary phytochemicals, resveratrol and curcumin, on aging and stress response.

Enhanced photostability of an anthracene-based dye due to supramolecular encapsulation: a new type of photostable fluorophore for single-molecule study.

For single-molecule fluorescence studies, highly photostable fluorophores are absolutely imperative, because photo-induced degradation (i.e., photobleaching) limits the observation time of individual molecules. Herein, the photophysics and photostability of a highly fluorescent 9,10-bis(phenylethynyl)anthracene derivative (G) and its self-assembled boronic ester encapsulation complex (G@Cap) embedded in a glassy polymer matrix are investigated by single-molecule fluorescence spectroscopy (SMFS). The heterogeneity of the fluorescence emission wavelength and triplet blinking kinetics of the guest G are significantly decreased by supramolecular encapsulation due to conformational restriction and reduced heterogeneity in the local environment. A nearly 10-fold increase in the photostability of G due to encapsulation is quantitatively confirmed by evaluating the photobleaching yields of G and G@Cap. In addition, it is found that the G@Cap is >30-fold more photostable than rhodamine 6G, a widely used fluorescent dye in single-molecule studies. These results demonstrate that the G@Cap can serve as a very bright, long-lasting fluorescent probe for single-molecule studies.

Determination of dehydroascorbic acid in mouse tissues and plasma by using tris(2-carboxyethyl)phosphine hydrochloride as reductant in metaphosphoric acid/ethylenediaminetetraacetic acid solution.

Ascorbic acid (AA) has a strong anti-oxidant function evident as its ability to scavenge superoxide radicals in vitro. Moreover, AA is an essential ingredient for post-translational proline hydroxylation of collagen molecules. Dehydroascorbic acid (DHA), the oxidized form of AA, is generated from these reactions. In this study, we describe an improved method for assessing DHA in biological samples. The use of 35 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP) as a reductant completely reduced DHA to AA after 2 h on ice in a 5% solution of metaphosphoric acid containing 1 mM ethylenediaminetetraacetic acid (EDTA) at pH 1.5. This method enabled us to measure the DHA content in multiple tissues and plasma of 6-weeks-old mice. The percentages of DHA per total AA differed markedly among these tissues, i.e., from 0.8 to 19.5%. The lung, heart, spleen and plasma had the highest levels at more than 10% of DHA per total AA content, whereas the cerebrum, cerebellum, liver, kidney and small intestine had less than 5% of DHA per total AA content. This difference in DHA content may indicate an important disparity of oxidative stress levels among physiologic sites. Therefore, this improved method provides a useful standard for all DHA determinations.

[Anti-aging studies on the senescence accelerated mouse (SAM) strains].

Senescence accelerated mouse (SAM), a murine model of accelerated senescence, was established by Toshio Takeda and colleagues. SAM consists of series of SAMP (prone) and SAMR (resistant) lines. All SAMP lines (from SAMP1 to SAMP11) are characterized by accelerated accumulation of senile features, earlier onset and faster progress of age-associated pathological phenotypes, such as amyloidosis, impaired immune response, senile osteoporosis and deficits in learning and memory. These SAMP lines are useful for evaluation of putative anti-aging therapies. For example, SAMP1 line is used to study the anti-aging effect of the antioxidant containing foods and various anti-oxidants, such as coenzyme Q10, vitamin C, lycopene. SAMP8 line exhibiting an early onset of impaired learning and memory is often used for test strategies for therapeutic intervention of dementia of early onset. SAMP6 is used as an animal model for developing new strategies for the treatment of osteoporosis in humans. Various lines of SAM (P1, P6, P8, P10 and R1) are now commercially available for research. In this review, I will briefly introduce various usages of SAM in anti-aging research.

Age-related difference of site-specific histone modifications in rat liver.

Aging is associated with decrease in activities of the transcription, replication and DNA repair that can result in deterioration of cellular and tissue functions. Changes of chromatin structures with age are likely major underling mechanisms for the functional decline. Chromatin consists of DNA and histones as well as non-histone proteins. While age-associated change of DNA methylation is well documented, little information is available on site-specific histone modifications in aging. We studied here age-related change of selected modifications of rat liver histone, i.e., histone H3 Lys9 acetylation (H3K9ac), H3 Lys9 methylation (H3K9me), H3 Ser10 phosphorylation (H3S10ph) and H3 Lys14 acetylation (H3K14ac). H3K9ac was decreased and H3S10ph was increased with age significantly. In view of reports indicating that decrease in acetylation and increase in phosphorylation of H3 histones can suppress gene activity, our findings suggest that a mechanism of decreased chromatin functions with age is due to such epigenetic changes.

Vitamin C is not essential for carnitine biosynthesis in vivo: verification in vitamin C-depleted senescence marker protein-30/gluconolactonase knockout mice.

Carnitine is an essential cofactor in the transport of long-chain fatty acids into the mitochondrial matrix and plays an important role in energy production via beta-oxidation. Vitamin C (VC) has long been considered a requirement for the activities of two enzymes in the carnitine biosynthetic pathway, i.e., 6-N-trimethyllysine dioxygenase and gamma-butyrobetaine dioxygenase. Our present study using senescence marker protein-30 (SMP30)/gluconolactonase (GNL) knockout (KO) mice, which cannot synthesize VC in vivo, led to the conclusion that this notion is not true. After weaning at 40 d of age, SMP30/GNL KO mice were fed a diet lacking VC and carnitine, then given water containing 1.5 g/l VC (VC(+) mice) or no VC (VC(-) mice) for 75 d. Subsequently, total VC and carnitine levels were measured in the cerebrum, cerebellum, liver, kidney, soleus muscle, extensor digitorum longus muscle, heart, plasma and serum. The total VC levels in all tissues and plasma from VC(-) SMP30/GNL KO mice were negligible, i.e., <2% of the levels in SMP30/GNL KO VC(+) mice; however, the total carnitine levels of both groups were similar in all tissues and serum. In addition, carnitine was produced by incubated liver homogenates from the VC-depleted SMP30/GNL KO mice irrespective of the presence or absence of 1 mM VC. Collectively, these results indicate that VC is not essential for carnitine biosynthesis in vivo.

Hydrogen-rich pure water prevents superoxide formation in brain slices of vitamin C-depleted SMP30/GNL knockout mice.

Hydrogen is an established anti-oxidant that prevents acute oxidative stress. To clarify the mechanism of hydrogen's effect in the brain, we administered hydrogen-rich pure water (H(2)) to senescence marker protein-30 (SMP30)/gluconolactonase (GNL) knockout (KO) mice, which cannot synthesize vitamin C (VC), also a well-known anti-oxidant. These KO mice were divided into three groups; recipients of H(2), VC, or pure water (H(2)O), administered for 33 days. VC levels in H(2) and H(2)O groups were <6% of those in the VC group. Subsequently, superoxide formation during hypoxia-reoxygenation treatment of brain slices from these groups was estimated by a real-time biography imaging system, which models living brain tissues, with Lucigenin used as chemiluminescence probe for superoxide. A significant 27.2% less superoxide formed in the H(2) group subjected to ischemia-reperfusion than in the H(2)O group. Thus hydrogen-rich pure water acts as an anti-oxidant in the brain slices and prevents superoxide formation.

Age-related change of endocytic receptors megalin and cubilin in the kidney in rats.

Megalin and cubilin are the major endocytic receptors responsible for resorption of glomerular filtrate proteins, particularly albumin, in the renal proximal tubule. In order to better understand the mechanism of the development of albuminuria with age in rats, we investigated age-related change of the amount and cellular localization of both receptors in the kidney. Immunoblot analysis of the kidney extracts showed that the amount of megalin significantly decreased with age. Although there was no age-related change in the amount of intact cubilin, the amount of cubilin fragments increased with age. Immunohistochemical study revealed that megalin and cubilin were predominantly localized in brush border membrane of proximal tubular cells in young rats, but the receptors tended to diffuse into the cytoplasm in the old rats. Interestingly, low but significant amounts of megalin and cubilin were present in the glomerular cells in addition to the proximal tubular cells. The quantity of receptors progressively increased in the glomerulus with age. This age-related increase might be to compensate for the age-related defect of the uptake of albumin by the proximal tubules. Thus, although it is unclear whether megalin and cubilin in the glomerulus contribute to the uptake of albumin in primary urine, the age-related increase in the amount of albumin in urine might at least partly be due to quantitative and qualitative alterations of both receptors in the proximal tubule.