Age-associated decline of MondoA drives cellular senescence through impaired autophagy and mitochondrial homeostasis.

Accumulation of senescent cells affects organismal aging and the prevalence of age-associated disease. Emerging evidence suggests that activation of autophagy protects against age-associated diseases and promotes longevity, but the roles and regulatory mechanisms of autophagy in cellular senescence are not well understood. Here, we identify the transcription factor, MondoA, as a regulator of cellular senescence, autophagy, and mitochondrial homeostasis. MondoA protects against cellular senescence by activating autophagy partly through the suppression of an autophagy-negative regulator, Rubicon. In addition, we identify peroxiredoxin 3 (Prdx3) as another downstream regulator of MondoA essential for mitochondrial homeostasis and autophagy. Rubicon and Prdx3 work independently to regulate senescence. Furthermore, we find that MondoA knockout mice have exacerbated senescence during ischemic acute kidney injury (AKI), and a decrease of MondoA in the nucleus is correlated with human aging and ischemic AKI. Our results suggest that decline of MondoA worsens senescence and age-associated disease.

Rapid multiplex PCR assay for simultaneous detection of Neisseria gonorrhoeae and Chlamydia trachomatis in genitourinary samples: A 30-minute assay.

We developed a rapid multiplex PCR assay available in bedside for the simultaneous detection of Neisseria gonorrhoeae and Chlamydia trachomatis, which enables diagnosis in less than 30 minutes. In this study, we validated the clinical utility of this assay including its sensitivity and specificity for NG and CT detection.

Trichloroethylene exposure aggravates behavioral abnormalities in mice that are deficient in superoxide dismutase.

Trichloroethylene (TCE) has been implicated as a causative agent for Parkinson's disease (PD). The administration of TCE to rodents induces neurotoxicity associated with dopaminergic neuron death, and evidence suggests that oxidative stress as a major player in the progression of PD. Here we report on TCE-induced behavioral abnormality in mice that are deficient in superoxide dismutase 1 (SOD1). Wild-type (WT) and SOD1-deficient (Sod1(-/-)) mice were intraperitoneally administered TCE (500 mg/kg) over a period of 4 weeks. Although the TCE-administrated Sod1(-/-) mice showed marked abnormal motor behavior, no significant differences were observed among the experimental groups by biochemical and histopathological analyses. However, treating mouse neuroblastoma-derived NB2a cells with TCE resulted in the down regulation of the SOD1 protein and elevated oxidative stress under conditions where SOD1 production was suppressed. Taken together, these data indicate that SOD1 plays a pivotal role in protecting motor neuron function against TCE toxicity.

Radical-scavenging Activity and Antioxidative Effects of Olive Leaf Components Oleuropein and Hydroxytyrosol in Comparison with Homovanillic Alcohol.

Olive leaf has great potential as a natural antioxidant, and one of its major phenolic components is oleuropein. In this study, the antioxidant activity of oleuropein against oxygen-centered radicals was measured by examining its sparing effects on the peroxyl radical-induced decay of fluorescein and pyrogallol red, in comparison with related compounds. The antioxidant capacity of oleuropein against lipid peroxidation was also assessed through its effect on the free radical-induced oxidation of methyl linoleate in a micelle system. On a molar basis, oleuropein and hydroxytyrosol inhibited the decay of fluorescein for longer than both homovanillic alcohol and the vitamin-E mimic 2-carboxy-2,5,7,8-tetramethyl-6-chromanol (Trolox), but did not suppress pyrogallol red decay in a concentration-dependent manner. Measurement of the fluorescein decay period revealed that the stoichiometric number of oleuropein and hydroxytyrosol against peroxyl radicals was twice that of Trolox, which is substantially higher than expectations based on chemical structure. Oleuropein and hydroxytyrosol were also more effective than Trolox and homovanillic alcohol at suppressing the oxidation of methyl linoleate in the micelle system. Thus, both oleuropein and hydroxytyrosol exhibit high antioxidative activity against lipid peroxidation induced by oxygen-centered radicals, but the high reactivity of phenolic/catecholic radicals makes their mechanism of action complex.

Isolation and characterization of antigen-specific alpaca (Lama pacos) VHH antibodies by biopanning followed by high-throughput sequencing.

The antigen-binding domain of camelid dimeric heavy chain antibodies, known as VHH or Nanobody, has much potential in pharmaceutical and industrial applications. To establish the isolation process of antigen-specific VHH, a VHH phage library was constructed with a diversity of 8.4 × 10(7) from cDNA of peripheral blood mononuclear cells of an alpaca (Lama pacos) immunized with a fragment of IZUMO1 (IZUMO1PFF) as a model antigen. By conventional biopanning, 13 antigen-specific VHHs were isolated. The amino acid sequences of these VHHs, designated as N-group VHHs, were very similar to each other (>93% identity). To find more diverse antibodies, we performed high-throughput sequencing (HTS) of VHH genes. By comparing the frequencies of each sequence between before and after biopanning, we found the sequences whose frequencies were increased by biopanning. The top 100 sequences of them were supplied for phylogenic tree analysis. In total 75% of them belonged to N-group VHHs, but the other were phylogenically apart from N-group VHHs (Non N-group). Two of three VHHs selected from non N-group VHHs showed sufficient antigen binding ability. These results suggested that biopanning followed by HTS provided a useful method for finding minor and diverse antigen-specific clones that could not be identified by conventional biopanning.

Heat-induced irreversible denaturation of the camelid single domain VHH antibody is governed by chemical modifications.

The variable domain of camelid heavy chain antibody (VHH) is highly heat-resistant and is therefore ideal for many applications. Although understanding the process of heat-induced irreversible denaturation is essential to improve the efficacy of VHH, its inactivation mechanism remains unclear. Here, we showed that chemical modifications predominantly governed the irreversible denaturation of VHH at high temperatures. After heat treatment, the activity of VHH was dependent only on the incubation time at 90 °C and was insensitive to the number of heating (90 °C)-cooling (20 °C) cycles, indicating a negligible role for folding/unfolding intermediates on permanent denaturation. The residual activity was independent of concentration; therefore, VHH lost its activity in a unimolecular manner, not by aggregation. A VHH mutant lacking Asn, which is susceptible to chemical modifications, had significantly higher heat resistance than did the wild-type protein, indicating the importance of chemical modifications to VHH denaturation.

Evaluation of biological activities of a groundnut (Apios americana Medik) extract containing a novel isoflavone.

Groundnut (Apios americana Medik) contains a novel isoflavone, genistein-7-O-gentiobioside. In the present study, we examined the biological activities of an alcohol extract of groundnut containing genistein-7-O-gentiobioside as the main component. Although the groundnut extract by itself did not show antioxidative activity, it drove the antioxidative system in cells. Pretreatment of human breast carcinoma MCF-7 cells for 24 h with the groundnut extract and soybean isoflavone increased gene expression of heme oxygenase-1 (HO-1), a major antioxidative stress enzyme. These groundnut extract-treated cells showed antioxidative activity against free radicals derived from a radical initiator. Pretreatment of cells with 100 µg/mL groundnut extract prevented the depletion of glutathione by the radical initiator; however, treatment with 100 µg/mL of soybean isoflavone injured the cell membrane, indicating that glutathione might be released to the extracellular environment. These results suggest that the groundnut extract had isoflavone-like activity. Like soybean, groundnuts are a good source of isoflavones.

Capacity of fucoxanthin for scavenging peroxyl radicals and inhibition of lipid peroxidation in model systems.

Carotenoids act as physiological antioxidant by scavenging reactive-free radicals as well as quenching singlet oxygen. Fucoxanthin is one of the abundant carotenoids found in edible brown seaweeds. The assessment of radical scavenging capacity of carotenoids has been the subject of extensive studies, which, however, gave inconsistent results. In the present study, the capacity of fucoxanthin for scavenging peroxyl radicals, chain carrying species of lipid peroxidation, was assessed quantitatively by measuring the effect of α-tocopherol on the decay of fucoxanthin induced by peroxyl radicals. It was found that α-tocopherol was 7.1 times more reactive than fucoxanthin in heptane solution, but interestingly fucoxanthin exerted 1.6 times higher reactivity than α-tocopherol in methanol solution. In SDS micelles, the relative reactivity of fucoxanthin and α-tocopherol depended on the site of peroxyl radical formation. The efficacy of lipid peroxidation inhibition by fucoxanthin was much less than that of α-tocopherol.

Capacity of peroxyl radical scavenging and inhibition of lipid peroxidation by ß-carotene, lycopene, and commercial tomato juice.

Carotenoids have received much attention as natural antioxidants. The role and action of carotenoids as singlet oxygen quenchers have been well demonstrated. The radical scavenging is another function of carotenoids as antioxidants, but the method to assess the radical scavenging capacity has not been established and previous studies have given inconsistent results. Carotenoids have strong absorptions in the visible light region and, unlike phenolic antioxidants, they do not have a reactive hydrogen to donate to radicals, which make it difficult to use conventional probes for the assessment of their radical scavenging capacity. In the present study, a method for the assessment of peroxyl radical scavenging capacity by carotenoids was re-examined and applied for ß-carotene, lycopene, and commercial tomato juice. The capacity for scavenging peroxyl radicals was assessed from the effect of α-tocopherol on the bleaching of carotenoids being tested. Total content of carotenoids was also assessed from the rate of bleaching under constant flux of peroxyl radicals. The peroxyl radical scavenging capacity of ß-carotene and lycopene exhibited was about one-tenth of that observed for α-tocopherol, and the efficacy of lipid peroxidation inhibition was much smaller.

Reactivity toward oxygen radicals and antioxidant action of thiol compounds.

Thiol compounds exert diverse functions in the defense network against oxidative stress in vivo. Above all, the role of glutathione in the enzymatic removal of hydrogen peroxide and lipid hydroperoxides has been well established. The scavenging of reactive free radicals is one of the many functions. In this study, the reactivities of several thiol compounds toward oxygen- and nitrogen-centered radicals were measured from their reaction with galvinoxyl and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals and also from their sparing effects on the decay of fluorescein, pyrogallol red, and BODIPY induced by peroxyl radicals. Furthermore, the antioxidant capacity against lipid peroxidation was assessed in the oxidation of methyl linoleate induced by free radicals in micelle systems. Cysteine, homocysteine, and glutathione exhibited considerable reactivity toward galvinoxyl, DPPH, and peroxyl radicals in this order but methionine did not. Bovine serum albumin (BSA) was less reactive toward these radicals than cysteine on molar base. Cysteine, homocysteine, and glutathione suppressed the oxidation of methyl linoleate in micelle systems, but methionine did not. The reactivity toward free radicals and antioxidant capacity of these thiol compounds were less than that of ascorbic acid, but higher than that of uric acid.

Assessment of antioxidant capacity for scavenging free radicals in vitro: a rational basis and practical application.

With increasing evidence showing the involvement of oxidative stress induced by free radicals in the development of various diseases, the role of radical-scavenging antioxidants has received much attention. Although many randomized controlled clinical trials do not support the beneficial effects of indiscriminate supplementation of antioxidants, more recent studies suggest that antioxidants such as vitamin E may be effective for prevention and treatment of some diseases when given to the right subjects at the right time. Many studies on the antioxidant capacity assessed by various available methods showed inconsistent results and the assessment of antioxidant capacity has been the subject of extensive studies and arguments. This study was performed to elucidate the basic chemistry required for the development of a reliable method for the assessment of antioxidant capacity for radical scavenging in vitro. In this study, the capacity of α-tocopherol and its related compounds, ascorbic acid, and uric acid for scavenging radicals was assessed from their effects on the rate of decay of hydrophilic and lipophilic probes with various reactivities toward free radicals induced by hydrophilic and lipophilic radicals in homogeneous solution and heterogeneous micelle systems. Fluorescein, pyranine, and pyrogallol red were used as hydrophilic probes, and BODIPY and N,N-diphenyl-p-phenylenediamine were used as lipophilic probes. We show that the rate and amount of radical scavenging by antioxidants, termed the antioxidant radical absorbance capacity, could be assessed by an appropriate combination of radical initiator and probe. This method was applied to the assessment of radical-scavenging capacity of human plasma, wine, and green tea powder.