Shuntaro Yamashita

SIRT1 prevents replicative senescence of normal human umbilical cord fibroblast through potentiating the transcription of human telomerase reverse transcriptase gene

SIRT1, the mammalian homolog of sirtuins, has emerged as a mediator of the beneficial effects of calorie restriction. Among them, we focused on the SIRT1-induced prevention of cellular senescence, and tried to reveal the molecular mechanisms that define the effects of SIRT1. Firstly in this study, we observed that overexpression of SIRT1 resulted in the prevention of cellular senescence of normal human umbilical cord fibroblast HUC-F2 cells. Here, we focused on the human telomerase reverse transcriptase (hTERT) gene as a target of the SIRT1-induced prevention of cellular senescence. Results showed that SIRT1, SIRT1 activator, resveratrol, and SIRT1 activating condition, starved condition, increased the transcription of hTERT in HUC-F2 cells. Next, we found that SIRT1 increased hTERT transcription in a c-MYC-dependent manner, triggered the transcription of the c-MYC gene and increased the amount of c-MYC recruited to the hTERT promoter. Further, SIRT1 increased the transcriptional activation ability of c-MYC and correspondingly increased the amount of acetylated H4 histone at the hTERT promoter. All of these results indicated that SIRT1 activates hTERT transcription through the involvement of c-MYC, and suggested that this SIRT1-induced augmentation of hTERT transcription resulted in the extension of the cellular life span of HUC-F2 cells.

Quantitative analysis of cellular senescence phenotypes using an imaging cytometer

Until now, various stimuli as well as serial passaging have been known to induce cellular senescence in normal human diploid fibroblasts. However, in many cases, we have encountered difficulty in quantitatively analyzing the cellular senescence phenotypes of senescent cells in a physiological condition. High-content screening (HCS)-based image analysis is becoming an important and powerful research tool. In the present study, an automated and quantitative cellular image-analysis system was employed to quantify the cellular senescence phenotypes induced in normal human diploid fibroblasts, TIG-1 cells, and found to be a powerful tool in the cellular senescence study.

FOXO3a Potentiates hTERT Gene Expression by Activating c-MYC and Extends the Replicative Life-Span of Human Fibroblast

In our previous studies, we reported that SIRT1 prevents cellular senescence in human fibroblast, and that SIRT1-induced inhibition of cellular senescence is due to enhanced hTERT gene expression. In this study, we investigate the molecular mechanisms behind SIRT1-induced potentiation of hTERT transcription and show that FOXO3a functions downstream of SIRT1 and prevents the induction of cellular senescence by enhancing hTERT gene expression. Furthermore, we found that FOXO3a-induced potentiation of hTERT gene expression is regulated in a c-MYC/E-box dependent manner. In addition, we found that FOXO3a binds to the novel binding element in the c-MYC promoter, and this interaction activates the transcription of the c-MYC gene. The resulting increase in c-MYC leads to higher levels of c-MYC recruited to the hTERT promoter and, in turn, activates hTERT gene expression. Taken together, this pathway might constitute the molecular basis for the anti-senescence effects of SIRT1 and FOXO3a.

Regulatory mechanisms of human and mouse telomerase reverse transcriptase gene transcription: distinct dependency on c-Myc

Telomerase—a complex ribonucleoprotein enzyme—synthesizes telomeric repeats to avoid telomere loss that accompanies cell division and chromosomal replication. Expression of telomerase is detectable in embryonic cells and cancer cells, but not in normal human cells. On the other hand, in mice, substantial expression of telomerase is detected in normal cells and tissues as well as in immortalized cells. These results suggest that the regulatory mechanisms of telomerase activity in humans and mice differ. Considering these results along with the fact that the expression of the telomerase reverse transcriptase (TERT) gene is a rate-limiting step for telomerase activity, we compared transcriptional regulatory mechanisms of both the species. A series of luciferase assays and RT-PCR analyses demonstrated that c-Myc, a dominant transactivator for human TERT (hTERT), is not involved in the regulation of mouse TERT (mTERT). These results suggest that distinct molecules and pathways are involved in the process of immortalization and tumorigenesis in human and mouse cells.

Novel purification method of human immunoglobulin by using a thermo-responsive protein A.

We attempted to evaluate a novel purification method of immunoglobulins (IgGs) by using a mutant type of protein A. Although this mutant protein A binds to IgGs at 5°C, the IgGs are released at 40°C; hence, it was designated as thermo-responsive protein A (TRPA). We aimed to purify IgG1 from the culture supernatant of CHO cells producing AE6F4 human monoclonal IgG1. AE6F4 IgG1 was purified using only a TRPA-filled column and by modifying the temperature, without any exposure to acidic conditions. Furthermore, the purified AE6F4 IgG1 maintained the inherent binding affinity to antigen, while this property was lost in AE6F4 IgG1 purified using a conventional protein A (CPA) column possibly because of product aggregation and fragmentation. These data suggest that IgG is sensitive to acid treatment; however, it can be highly purified with retention of high affinity by using a TRPA column. Further, this purification method can be used on an industrial scale for the purification of antibody drugs.

In vitro neuroprotective activities of compounds from Angelica shikokiana Makino

Angelica shikokiana is widely marketed in Japan as a dietary food supplement. With a focus on neurodegenerative conditions such as Alzheimer’s disease, the aerial part was extracted and through bio-guided fractionation, fifteen compounds [α-glutinol, β-amyrin, kaempferol, luteolin, quercetin, kaempferol-3-O-glucoside, kaempferol-3-O-rutinoside, methyl chlorogenate, chlorogenic acid, hyuganin E, 5-(hydroxymethyl)-2-furaldehyde, β-sitosterol-3-O-glucoside, adenosine (isolated for the first time from A. shikokiana), isoepoxypteryxin and isopteryxin] were isolated. Isolated compounds were evaluated for in vitro neuroprotection using acetylcholine esterase inhibitory, protection against hydrogen peroxide and amyloid β peptide (Aβ25-35)-induced neurotoxicity in neuro-2A cells, scavenging of hydroxyl radicals and intracellular reactive oxygen species and thioflavin T assays. Quercetin showed the strongest AChE inhibition (IC50 value = 35.5 µM) through binding to His-440 and Tyr-70 residues at the catalytic and anionic sites of acetylcholine esterase, respectively. Chlorogenic acid, its methyl ester, quercetin and luteolin could significantly protect neuro-2A cells against H2O2-induced neurotoxicity and scavenge hydroxyl radical and intracellular reactive oxygen species. Kaempferol-3-O-rutinoiside, hyuganin E and isoepoxypteryxin significantly decreased Aβ25-35-induced neurotoxicity and Th-T fluorescence. To the best of our knowledge, this is the first report about neuroprotection of hyuganin E and isoepoxypteryxin against Aβ25-35-induced neurotoxicity.

Wild Mushrooms in Nepal: Some Potential Candidates as Antioxidant and ACE-Inhibition Sources

Twenty-nine mushrooms collected in the mountainous areas of Nepal were analyzed for antioxidant activity by different methods, including Folin-Ciocalteu, ORAC, ABTS, and DPPH assays. Intracellular H2O2-scavenging activity was also performed on HaCaT cells. The results showed that phenolic compounds are the main antioxidant of the mushrooms. Among studied samples, Inonotus andersonii, and Phellinus gilvus exhibited very high antioxidant activity with the phenolic contents up to 310.8 and 258.7 mg GAE/g extracts, respectively. The H2O2-scavenging assay on cells also revealed the potential of these mushrooms in the prevention of oxidative stress. In term of ACE-inhibition, results showed that Phlebia tremellosa would be a novel and promising candidate for antihypertensive studies. This mushroom exhibited even higher in vitro ACE-inhibition activity than Ganoderma lingzhi, with the IC50 values of the two mushrooms being 32 μg/mL and 2 μg/mL, respectively. This is the first time biological activities of mushrooms collected in Nepal were reported. Information from this study should be a valuable reference for future studies on antioxidant and ACE-inhibitory activities of mushrooms.

Mechanisms of carnosine-induced activation of neuronal cells

Abstract Carnosine (β-Ala-l-His), an imidazole dipeptide, is known to have many functions. Recently, we demonstrated in a double-blind randomized controlled trial that carnosine is capable of preserving cognitive function in elderly people. In the current study, we assessed the ability of carnosine to activate the brain, and we tried to clarify the molecular mechanisms behind this activation. Our results demonstrate that carnosine permeates the blood brain barrier and activates glial cells within the brain, causing them to secrete neurotrophins, including BDNF and NGF. These results point to a novel mechanism of carnosine-induced neuronal activation. Our results suggest that carnosine should be recognized as a functional food factor that helps achieve anti-brain aging. Graphical abstract Schematic diagram of carnosine function in the brain.

Mushroom extract inhibits ultraviolet B-induced cellular senescence in human keratinocytes

Mushrooms possess various bioactivities and are used as nutritional supplements and medicinal products. Twenty-nine bioactive components have been extracted recently from mushrooms grown in Nepal. In this study, we evaluated the ability of these mushroom extracts to augment SIRT1, a mammalian SIR2 homologue localized in cytosol and nuclei. We established a system for screening food ingredients that augment the SIRT1 promoter in HaCaT cells, and identified a SIRT1-augmenting mushroom extract (number 28, Trametes versicolor). UVB irradiation induced cellular senescence in HaCaT cells, as evidenced by increased activity and expression of cellular senescence markers including senescence-associated β-galactosidase, p21, p16, phosphorylated p38, and γH2AX. Results clearly showed that the mushroom extract (No. 28) suppressed the ultraviolet B irradiation-induced cellular senescence in HaCaT cells possibly through augmenting SIRT1 expression.