Improving lysosomal ferroptosis with NMN administration protects against heart failure.

Myocardial mitochondria are primary sites of myocardial energy metabolism. Mitochondrial disorders are associated with various cardiac diseases. We previously showed that mice with cardiomyocyte-specific knockout of the mitochondrial translation factor p32 developed heart failure from dilated cardiomyopathy. Mitochondrial translation defects cause not only mitochondrial dysfunction but also decreased nicotinamide adenine dinucleotide (NAD+) levels, leading to impaired lysosomal acidification and autophagy. In this study, we investigated whether nicotinamide mononucleotide (NMN) administration, which compensates for decreased NAD+ levels, improves heart failure because of mitochondrial dysfunction. NMN administration reduced damaged lysosomes and improved autophagy, thereby reducing heart failure and extending the lifespan in p32cKO mice. We found that lysosomal damage due to mitochondrial dysfunction induced ferroptosis, involving the accumulation of iron in lysosomes and lipid peroxide. The ameliorative effects of NMN supplementation were found to strongly affect lysosomal function rather than mitochondrial function, particularly lysosome-mediated ferroptosis. NMN supplementation can improve lysosomal, rather than mitochondrial, function and prevent chronic heart failure.

Prominent Prolongation of Cortical Silent Period Induced by Transcranial Magnetic Stimulation in Creutzfeldt-Jakob Disease.

The cortical silent period (CSP) induced by transcranial magnetic stimulation (TMS) has been reported to be prolonged in 2 Creutzfeldt-Jakob disease (CJD) patients who presented with periodic myoclonus. Herein, we will show a prominent prolongation of TMS-induced CSP in a patient with CJD who did not have periodic myoclonus. The patient was a 66-year-old woman who developed rapidly progressive dementia. No myoclonic jerks were observed. Brain magnetic resonance imaging showed high-intensity lesions in the cerebral cortex, basal ganglia, and thalamus on diffusion-weighted images. Electroencephalography (EEG) showed diffuse and continuous slow waves, but no periodic synchronous discharges (PSDs). A TMS study revealed that the duration of CSP was prominently prolonged: the duration of CSP (370 ms) equaled that of the mean + 6.5 SD of the normal value. One month after admission, the patient exhibited akinetic mutism and developed periodic myoclonus in her limbs. The clinical course was compatible with CJD. To date, CSP has been measured in only 2 CJD patients. The common findings in both cases were marked prolongation of CSP, periodic myoclonus, and PSD on EEG. In short, we demonstrated that TMS-induced CSP was prominently prolonged even at the early stage of CJD without periodic myoclonus or PSD. In other disorders, the CSP has not been reported to be comparably prolonged to that of CJD patients. Therefore, we conclude that TMS-induced CSP could be prominently prolonged even in the early stage of CJD. The marked prolongation of the CSP might be an early biomarker of CJD.

Simple method for large-scale production of macrophage activating factor GcMAF.

Human group-specific component protein (Gc protein) is a multifunctional serum protein which has three common allelic variants, Gc1F, Gc1S and Gc2 in humans. Gc1 contains an O-linked trisaccharide [sialic acid-galactose-N-acetylgalactosamine (GalNAc)] on the threonine420 (Thr420) residue and can be converted to a potent macrophage activating factor (GcMAF) by selective removal of sialic acid and galactose, leaving GalNAc at Thr420. In contrast, Gc2 is not glycosylated. GcMAF is considered a promising candidate for immunotherapy and antiangiogenic therapy of cancers and has attracted great interest, but it remains difficult to compare findings among research groups because different procedures have been used to prepare GcMAF. Here, we present a simple, practical method to prepare high-quality GcMAF by overexpressing Gc-protein in a serum-free suspension culture of ExpiCHO-S cells, without the need for a de-glycosylation step. We believe this protocol is suitable for large-scale production of GcMAF for functional analysis and clinical testing.

In vivo drug screening method of radiosensitizers using tumor-bearing chick embryo.

In radiotherapy, tumor hypoxia is the main factor responsible for treatment resistance, and the development of radiosensitizers that can overcome this is imperative. However, many drugs that are effective in vitro and in vivo fail in clinical trials, and thus it is necessary to develop an animal model that can be used for the correct evaluation of pharmacokinetics and activity. Developing chicken eggs are commonly used in various research fields such as anticancer drug sensitivity tests and cardiotoxicity tests. We examined whether the radiosensitizing activity of etanidazole, as a hypoxic cell radiosensitizer, could be evaluated using tumor-bearing chick embryo. Following the transplantation of mouse mammary carcinoma EMT6 cells on day 11, a solid tumor was formed on day 15 and an evaluation of the time-course of the tumor revealed that the tumor weight was the highest on day 18. The maximum dose of etanidazole that did not affect tumor growth and fetal survival was 1.0mg and the maximum X-ray dose was 8Gy. Etanidazole was intravenously administered 10min prior to single dose X-ray irradiation. A significant tumor growth inhibitory effect was confirmed with 1.0mg of etanidazole in combination with 8Gy X-ray. In the case of mouse colon cancer colon26 cells, the combination of 3.0mg of etanidazole and 2Gy X-ray showed 2.79 times higher radiosensitizing activity than that observed for the control group. These results demonstrate that it is possible to evaluate the activity of radiosensitizers using tumor-bearing chick embryo.

Extraterrestrial ribose and other sugars in primitive meteorites.

Sugars are essential molecules for all terrestrial biota working in many biological processes. Ribose is particularly essential as a building block of RNA, which could have both stored information and catalyzed reactions in primitive life on Earth. Meteorites contain a number of organic compounds including key building blocks of life, i.e., amino acids, nucleobases, and phosphate. An amino acid has also been identified in a cometary sample. However, the presence of extraterrestrial bioimportant sugars remains unclear. We analyzed sugars in 3 carbonaceous chondrites and show evidence of extraterrestrial ribose and other bioessential sugars in primitive meteorites. The 13C-enriched stable carbon isotope compositions (δ13C vs.VPDB) of the detected sugars show that the sugars are of extraterrestrial origin. We also conducted a laboratory simulation experiment of a potential sugar formation reaction in space. The compositions of pentoses in meteorites and the composition of the products of the laboratory simulation suggest that meteoritic sugars were formed by formose-like processes. The mineral compositions of these meteorites further suggest the formation of these sugars both before and after the accretion of their parent asteroids. Meteorites were carriers of prebiotic organic molecules to the early Earth; thus, the detection of extraterrestrial sugars in meteorites establishes the existence of natural geological routes to make and preserve them as well as raising the possibility that extraterrestrial sugars contributed to forming functional biopolymers like RNA on the early Earth or other primitive worlds.

Development of a novel Schiff base derivative for enhancing the anticancer potential of 5-aminolevulinic acid-based photodynamic therapy.

5-Aminolevulinic acid (ALA), a precursor of protoporphyrin IX (PpIX), is now widely used for photodynamic diagnosis (ALA-PDD) and photodynamic therapy (ALA-PDT) of various cancers. Recently, we found that treatment of cancer cells with the Schiff base derivative TX-816 along with ALA could significantly increase the efficacy of ALA-PDT. This enhancing effect of TX-816 on ALA-PDT is attributed to 3,5-dichlorosalicylaldehyde (DCSA), a molecule produced by the degradation of TX-816. Similar to TX-816, DCSA significantly enhances the effect of ALA-PDT. Furthermore, DCSA could restore the sensitivity of cancer cells that acquired resistance to ALA-PDT. These results indicate that DCSA, as well as TX-816, is a potent lead compound for the development of an ALA-PDT sensitizer. TX-816 might be a useful compound for designing prodrug-type ALA-PDT enhancers.

Establishment of a highly sensitive sandwich ELISA for the N-terminal fragment of titin in urine.

Muscle damage and loss of muscle mass are triggered by immobilization, loss of appetite, dystrophies and chronic wasting diseases. In addition, physical exercise causes muscle damage. In damaged muscle, the N-terminal and C-terminal regions of titin, a giant sarcomere protein, are cleaved by calpain-3, and the resulting fragments are excreted into the urine via glomerular filtration. Therefore, we considered titin fragments as promising candidates for reliable and non-invasive biomarkers of muscle injury. Here, we established a sandwich ELISA that can measure the titin N-terminal fragment over a biologically relevant range of concentrations, including those in urine samples from older, non-ambulatory Duchenne muscular dystrophy patients and from healthy donors under everyday life conditions and after exercise. Our results indicate that the established ELISA could be a useful tool for the screening of muscular dystrophies and also for monitoring the progression of muscle disease, evaluating the efficacy of therapeutic approaches, and investigating exercise-related sarcomeric disruption and repair processes.

Evaluation of the in vivo antioxidative activity of redox nanoparticles by using a developing chicken egg as an alternative animal model.

Antioxidants have been demonstrated to exert beneficial effects as pharmacotherapies for cardiovascular diseases. The in vitro systems generally employed to evaluate antioxidants, however, are limited by having no appreciable in vivo redox status of the antioxidants. Therefore, we used our developing chicken egg model to evaluate the in vivo antioxidative activity of a redox nanoparticle possessing 2,2,6,6-tetramethylpiperidine-1-oxyl (RNP(O)). The 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH) elicited strong oxidative stress and its LD50 value for chick embryos was 3.5±0.9mg/egg. The low molecular weight nitroxide compound, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL), which is known to have the highest level of antioxidant activity, showed no significant protective effect against AAPH-induced embryo lethality. On the contrary, RNP(O) had potent protective effects against AAPH-induced embryo lethality. Moreover, RNP(O) could significantly suppress the production of lipid peroxides in chick serum induced by hydrocortisone. Since RNP(O) has a longer retention time in blood than TEMPOL, RNP(O) may protect the embryo against lethal oxidative stress by suppressing lipid peroxidation. The validity of in vivo experiments using developing chicken eggs was supported by our data, where RNP(O) was determined to elicit strong antioxidative activity in vivo, irrespective of the lack of a significant difference in the in vitro activity between low-molecular weight TEMPOL and RNP(O). Our results support the use of the developing chicken egg model to evaluate the potential in vivo antioxidative activity of RNP(O).

Degalactosylated/desialylated human serum containing GcMAF induces macrophage phagocytic activity and in vivo antitumor activity.

BACKGROUND: The group-specific component protein-derived macrophage-activating factor (GcMAF) has various biological activities, such as macrophage activation and antitumor activity. Clinical trials of GcMAF have been carried out for metastatic breast cancer, prostate cancer, and metastatic colorectal cancer. In this study, despite the complicated purification process of GcMAF, we used enzymatically-treated human serum containing GcMAF with a considerable macrophage-stimulating activity and antitumor activity. RESULTS: We detected GcMAF in degalactosylated/desialylated human serum by western blotting using an anti-human Gc globulin antibody, and Helix pomatia agglutinin lectin. We also found that GcMAF-containing human serum significantly enhanced the phagocytic activity of mouse peritoneal macrophages and extended the survival time of mice bearing Ehrlich ascites tumors. CONCLUSION: We demonstrated that GcMAF-containing human serum can be used as a potential macrophage activator for cancer immunotherapy.

Evaluation of the in vivo radiosensitizing activity of etanidazole using tumor-bearing chick embryo.

Chick embryos have been used as alternative experimental animals in various research fields, including virology, immunology, toxicology, oncology, and embryology. Until now, there have been no in vivo models using chick embryo to evaluate radiosensitizing activity. Here, the in vivo radiosensitizing activity of etanidazole, a well-known hypoxic cell radiosensitizer, was evaluated using tumor-bearing chick embryo. On the basis of tumor growth, drug administration and X-ray irradiation were performed on day 15 chick embryo, with the endpoint being day 18 chick embryo. In day 15 chick embryo, an X-ray irradiation dose of equal or less than 10 Gy did not cause significant tumor growth suppression. Intravenous administration of equal or less than 1.0 mg of etanidazole did not cause tumor growth suppression. Neither doses of equal or less than 8 Gy of irradiation nor 1.0 mg of etanidazole caused fatality of the chick embryo. On the basis of these results, we evaluated the radiosensitizing effect of a combination treatment with 8 Gy of irradiation and 1.0 mg of etanidazole. As noted above, 1.0 mg of etanidazole alone and 8 Gy of irradiation alone did not show tumor growth suppression. In contrast, a combination treatment with 8 Gy of irradiation and 1.0 mg of etanidazole showed 35% of significant tumor growth suppression. Thus, we succeeded in evaluating the in vivo radiosensitizing activity of etanidazole using tumor-bearing chick embryo. These results suggest that the use of tumor-bearing chick embryo may be part of a promising system for evaluating radiosensitizing activity.

Design of a bioreductively-activated fluorescent pH probe for tumor hypoxia imaging.

We have designed and evaluated UTX-12 as a novel fluorescent pH probe for tumor hypoxia imaging. UTX-12 consists of a p-nitro benzyl moiety, which is a latent hypoxia-selective leaving group activated by nitro reduction, directly linked to SNARF. Although UTX-12 itself is colorless and non-fluorescent in aqueous solution, nitro reduction triggers the release of SNARF which has well-characterized long wavelength absorption and fluorescence that is sensitive to pH. The resultant SNARF, released intracellularly by enzymatic reduction of UTX-12, allows measurement of pH by pH-dependent dual emission shifts. UTX-12 showed clear differences in fluorescence behavior between hypoxic and aerobic conditions in liver microsomes and inside V79 cells. These data are confirmation that UTX-12 is biologically reduced inside tumor cells and the released SNARF should monitor intracellular pH of tumor cells selectively with reduced background signal.

A chemical biosynthesis design for an antiatherosclerosis drug by acyclic tocopherol intermediate analogue based on "isoprenomics".

Phytyl quinols, namely acyclic tocopherols, are key intermediates of tocopherol biosynthesis, but their biological activities remain unclear. We therefore investigated the structure-activity relationship of phytyl quinols to apply a chemical biosynthesis design for an antiatherosclerosis drug based on isoprenomics. We have achieved the biosynthesis-oriented design and synthesis of alpha- (TX-2254) and beta-(TX-2247) phytyl quinol as an unnatural intermediate, other gamma- (TX-2242) and delta-(TX-2231) phytyl quinol as a natural one. Geometry optimization and Molecular orbital (MO) calculation of TX-2254 showed a unique right-angle structure; however, MO energy of TX-2254 and d-alpha-tocopherol were very similar. Radical reactivity of TX-2231 was equal to dl-alpha-tocopherol, whereas TX-2254, TX-2247, and TX-2231 showed lower reactivity than dl-alpha-tocopherol. All four phytyl quinols showed almost the same moderate inhibitory activity against low-density lipoprotein (LDL) oxidation instead of their different degree of C-methylation with character different from tocopherols. In vivo toxicities of phytyl quinols against chick embryo chorioallantoic membrane (CAM) vasculature were hardly observed. We proposed phytyl quinols were possible antioxidants in plants and animals, like vitamin E.