Validation of a Dehydroepiandrosterone-Sulfate Assay in Three Platyrrhine Primates (Alouatta caraya,Aotus azarae infulatus,and Sapajus apella)

International Journal of Primatology - The hormone dehydroepiandrosterone (DHEA) and its sulfate ester (DHEAS) are the most abundant circulating steroids in human and some nonhuman primates, and...     

Parkin-independent mitophagy via Drp1-mediated outer membrane severing and inner membrane ubiquitination

Here, we report that acute reduction in mitochondrial translation fidelity (MTF) causes ubiquitination of the inner mitochondrial membrane (IMM) proteins, including TRAP1 and CPOX, which occurs selectively in mitochondria with a severed outer mitochondrial membrane (OMM). Ubiquitinated IMM recruits the autophagy machinery. Inhibiting autophagy leads to increased accumulation of mitochondria with severed OMM and ubiquitinated IMM. This process occurs downstream of the accumulation of cytochrome c/CPOX in a subset of mitochondria heterogeneously distributed throughout the cell (“mosaic distribution”). Formation of mosaic mitochondria, OMM severing, and IMM ubiquitination require active mitochondrial translation and mitochondrial fission, but not the proapoptotic proteins Bax and Bak. In contrast, in Parkin-overexpressing cells, MTF reduction does not lead to the severing of the OMM or IMM ubiquitination, but it does induce Drp1-independent ubiquitination of the OMM. Furthermore, high–cytochrome c/CPOX mitochondria are preferentially targeted by Parkin, indicating that in the context of reduced MTF, they are mitophagy intermediates regardless of Parkin expression. In sum, Parkin-deficient cells adapt to mitochondrial proteotoxicity through a Drp1-mediated mechanism that involves the severing of the OMM and autophagy targeting ubiquitinated IMM proteins.     

Enhanced skeletal muscle glycogen repletion after endurance exercise is associated with higher plasma insulin and skeletal muscle hexokinase 2 protein levels in mice: comparison of level running and downhill running model

Journal of Physiology and Biochemistry - To identify factors that influence post-exercise muscle glycogen repletion, we compared the glycogen recovery after level running with downhill running, an...     

Hydroxyl Radical Generation Caused by the Reaction of Singlet Oxygen with a Spin Trap,DMPO, Increases Significantly in the Presence of Biological Reductants

Photosensitizers newly developed for photodynamic therapy of cancer need to be assessed using accurate methods of measuring reactive oxygen species (ROS). Little is known about the characteristics of the reaction of singlet oxygen (¹O²) with spin traps, although this knowledge is necessary in electron spin resonance (ESR)/spin trapping. In the present study, we examined the effect of various reductants usually present in biological samples on the reaction of ¹O² with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The ESR signal of the hydroxyl radical (?OH) adduct of DMPO (DMPO-OH) resulting from ¹O²-dependent generation of ?OH strengthened remarkably in the presence of reduced glutathione (GSH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), ascorbic acid, NADPH, etc. A similar increase was observed in the photosensitization of uroporphyrin (UP), rose bengal (RB) or methylene blue (MB). Use of 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO) as a spin trap significantly lessened the production of its ?OH adduct (DEPMPO-OH) in the presence of the reductants. The addition of DMPO to the DEPMPO-spin trapping system remarkably increased the signal intensity of DEPMPO-OH. DMPO-mediated generation of ?OH was also confirmed utilizing the hydroxylation of salicylic acid (SA). These results suggest that biological reductants enhance the ESR signal of DMPO-OH produced by DMPO-mediated generation of ?OH from ¹O², and that spin trap-mediated ?OH generation hardly occurs with DEPMPO.     

A Novel Method for the Synthesis of ddA and F-ddA Via Regioselective 2′-O-Deacetylation of 9-(2,5-DI-O-Acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)adenine

Abstract

Regioselective 2′-O-deacetylation of 9-(2,5-di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)adenine (1) is achieved by treatment of 1 with β-cyclodextrin (β-CyD) / aq. NaHCO₃ or N₂H₄·H₂O / EtOH. The 9-(5-O-Acetyl-3-bromo-3-deoxy-β-D-xylo-furanosyl)adenine (2) obtained is a common intermediate for the synthesis of 2′,3′-dideoxy-adenosine (ddA) (7) and 9-(2-fluoro-2,3-dideoxy-β-D-threo-pentofuranosyl)-adenine (F-ddA) (9).     

Anesthetic Effects of a Mixture of Medetomidine,Midazolam and Butorphanol in Two Strains of Mice

The combination of ketamine and xylazine is a widely used anesthetic for laboratory animals. However, due to an abuse problem in Japan, ketamine has been specified as a narcotic since 2007. Instead of using ketamine, Kawai et al. reported an injectable formula with an equivalent effect to the mixture of ketamine and xylazine [11]. The mixture of 0.3 mg/kg body weight (b.w.) medetomidine (Med.), 4.0 mg/kg b.w. midazoram (Mid.), and 5.0 mg/kg b.w. butorphanol (But.) produced an anesthetic duration of around 40 min in outbred ICR mice. However, the anesthetic effect of the mixture for inbred mice strains remains unknown. Therefore, we examined anesthetic effects of the mixture of Med., Mid., and But. in the BALB/c and C57BL/6J strains. After intraperitoneal injection into mice, right front paw, left hind paw, and tail pinch reflexes as well as corneal and righting reflexes were observed. Every 5 min, we scored each reflex category as 0 for reaction or 1 for no reaction. As long as the total score was at least 4 out of 5, we considered the mixture as putting a mouse in a surgical anesthetic state. The mixture produced an anesthetic duration of more than 45 min in both strains of mice. These results indicate that the mixture of Med., Mid., and But. can be a useful and effective anesthesia for the BALB/c and C57BL/6J strains of inbred mice as well as outbred ICR mice.     

Selenoneine, a Novel Selenium-Containing Compound, Mediates Detoxification Mechanisms against Methylmercury Accumulation and Toxicity in Zebrafish Embryo

The selenium (Se)-containing antioxidant selenoneine (2-selenyl-N _α,N _α,N _α-trimethyl-l-histidine) has recently been discovered to be the predominant form of organic Se in tuna blood. Although dietary intake of fish Se has been suggested to reduce methylmercury (MeHg) toxicity, the molecular mechanism of MeHg detoxification by Se has not yet been determined. Here, we report evidence that selenoneine accelerates the excretion and demethylation of MeHg, mediated by a selenoneine-specific transporter, organic cations/carnitine transporter-1 (OCTN1). Selenoneine was incorporated into human embryonic kidney HEK293 cells transiently overexpressing OCTN1 and zebrafish blood cells by OCTN1. The K _m for selenoneine uptake was 13.0 μM in OCTN1-overexpressing HEK293 cells and 9.5 μM in zebrafish blood cells, indicating high affinity of OCTN1 for selenoneine in human and zebrafish cells. When such OCTN1-expressing cells and embryos were exposed to MeHg–cysteine (MeHgCys), MeHg accumulation was decreased and the excretion and demethylation of MeHg were enhanced by selenoneine. In addition, exosomal secretion vesicles were detected in the culture water of embryos that had been microinjected with MeHgCys, suggesting that these may be responsible for MeHg excretion and demethylation. In contrast, OCTN1-deficient embryos accumulated MeHg, and MeHg excretion and demethylation were decreased. Furthermore, Hg accumulation was decreased in OCTN1-overexpressing HEK293 cells, but not in mock vector-transfected cells, indicating that selenoneine and OCTN1 can regulate MeHg detoxification in human cells. Thus, the selenoneine-mediated OCTN1 system regulates secretory lysosomal vesicle formation and MeHg demethylation.     

Conformational Analysis of Isolated Domains of Helicobacter pylori CagA

The CagA protein of Helicobacter pylori is associated with increased virulence and gastric cancer risk. CagA is translocated into the host cell by a H. pylori type IV secretion system via mechanisms that are poorly understood. Translocated CagA interacts with numerous host factors, altering a variety of host signalling pathways. The recently determined crystal structure of C-terminally-truncated CagA indicated the presence of two domains: the smaller, flexible N-terminal domain and the larger, middle domain. In this study, we have investigated the conformation, oligomeric state and stability of the N-terminal, middle and glutamate-proline-isoleucine-tyrosine-alanine (EPIYA)-repeats domains. All three domains are monomeric, suggesting that the multimerisation of CagA observed in infected cells is likely to be mediated not by CagA itself but by its interacting partners. The middle and the C-terminal domains, but not the N-terminal domain, are capable of refolding spontaneously upon heat denaturation, lending support to the hypothesis that unfolded CagA is threaded C-terminus first through the type IV secretion channel with its N-terminal domain, which likely requires interactions with other domains to refold, being threaded last. Our findings also revealed that the C-terminal EPIYA-repeats domain of CagA exists in an intrinsically disordered premolten globule state with regions in PPII conformation - a feature that is shared by many scaffold proteins that bind multiple protein components of signalling pathways. Taken together, these results provide a deeper understanding of the physicochemical properties of CagA that underpin its complex cellular and oncogenic functions.     

Autologous Bone-Marrow Mesenchymal Stem Cell Implantation and Endothelial Function in a Rabbit Ischemic Limb Model

Background

The purpose of this study was to determine whether autologous mesenchymal stem cells (MSCs) implantation improves endothelial dysfunction in a rabbit ischemic limb model.

Methods

We evaluated the effect of MSC implantation on limb blood flow (LBF) responses to acetylcholine (ACh), an endothelium-dependent vasodilator, and sodium nitroprusside (SNP), an endothelium-independent vasodilator, in rabbits with limb ischemia in which cultured MSCs were implanted (n = 20) or saline was injected as a control group (n = 20). LBF was measured using an electromagnetic flowmeter. A total of 10⁶ MSCs were implanted into each ischemic limb.

Results

Histological sections of ischemic muscle showed that capillary index (capillary/muscle fiber) was greater in the MSC implantation group than in the control group. Laser Doppler blood perfusion index was significantly increased in the MSC implantation group compared with that in the control group. LBF response to ACh was greater in the MSC group than in the control group. After administration of N^G-nitro-L-arginine, a nitric oxide synthase inhibitor, LBF response to ACh was similar in the MSC implantation group and control group. Vasodilatory effects of SNP in the two groups were similar.

Conclusions

These findings suggest that MSC implantation induces angiogenesis and augments endothelium-dependent vasodilation in a rabbit ischemic model through an increase in nitric oxide production.