Colocalization of 14-3-3 proteins with SOD1 in Lewy body-like hyaline inclusions in familial amyotrophic lateral sclerosis cases and the animal model

Background and Purpose

Cu/Zn superoxide dismutase (SOD1) is a major component of Lewy body-like hyaline inclusion (LBHI) found in the postmortem tissue of SOD1-linked familial amyotrophic lateral sclerosis (FALS) patients. In our recent studies, 14-3-3 proteins have been found in the ubiquitinated inclusions inside the anterior horn cells of spinal cords with sporadic amyotrophic lateral sclerosis (ALS). To further investigate the role of 14-3-3 proteins in ALS, we performed immunohistochemical analysis of 14-3-3 proteins and compared their distributions with those of SOD1 in FALS patients and SOD1-overexpressing mice.

Methods

We examined the postmortem brains and the spinal cords of three FALS cases (A4V SOD1 mutant). Transgenic mice expressing the G93A mutant human SOD1 (mutant SOD1-Tg mice), transgenic mice expressing the wild-type human SOD1 (wild-type SOD1-Tg mice), and non-Tg wild-type mice were also subjected to the immunohistochemical analysis.

Results

In all the FALS patients, LBHIs were observed in the cytoplasm of the anterior horn cells, and these inclusions were immunopositive intensely for pan 14-3-3, 14-3-3β, and 14-3-3γ. In the mutant SOD1-Tg mice, a high degree of immunoreactivity for misfolded SOD1 (C4F6) was observed in the cytoplasm, with an even greater degree of immunoreactivity present in the cytoplasmic aggregates of the anterior horn cells in the lumbar spinal cord. Furthermore, we have found increased 14-3-3β and 14-3-3γ immunoreactivities in the mutant SOD1-Tg mice. Double immunofluorescent staining showed that C4F6 and 14-3-3 proteins were partially co-localized in the spinal cord with FALS and the mutant SOD1-Tg mice. In comparison, the wild-type SOD1-Tg and non-Tg wild-type mice showed no or faint immunoreactivity for C4F6 and 14-3-3 proteins (pan 14-3-3, 14-3-3β, and 14-3-3γ) in any neuronal compartments.

Discussion

These results suggest that 14-3-3 proteins may be associated with the formation of SOD1-containing inclusions, in FALS patients and the mutant SOD1-Tg mice.     

Eye gaze during observation of static faces in deaf people

Knowing where people look when viewing faces provides an objective measure into the part of information entering the visual system as well as into the cognitive strategy involved in facial perception. In the present study, we recorded the eye movements of 20 congenitally deaf (10 male and 10 female) and 23 (11 male and 12 female) normal-hearing Japanese participants while they evaluated the emotional valence of static face stimuli. While no difference was found in the evaluation scores, the eye movements during facial observations differed among participant groups. The deaf group looked at the eyes more frequently and for longer duration than the nose whereas the hearing group focused on the nose (or the central region of face) more than the eyes. These results suggest that the strategy employed to extract visual information when viewing static faces may differ between deaf and hearing people.     

Brain expression level and activity of HDAC6 protein in neurodegenerative dementia

Histone deacetylase 6 (HDAC6) is a multifunctional cytoplasmic protein that plays an especially critical role in the formation of aggresomes, where aggregates of excess protein are deposited. Previous immunohistochemical studies have shown that HDAC6 accumulates in Lewy bodies in Parkinson’s disease and dementia with Lewy bodies (DLB) as well as in glial cytoplasmic inclusions in multiple system atrophy (MSA). However, it is uncertain whether the level and activity of HDAC6 are altered in the brains of patients with neurodegenerative dementia. In the present study, we demonstrated that the level of HDAC6 was not altered in the temporal cortex of patients with Alzheimer’s disease and DLB in comparison with controls. In contrast, the level of HDAC6 was significantly increased in the temporal cortex of patients with frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) and in the cerebellar white matter of patients with MSA. However, the level of acetylated α-tubulin, one of the substrates of HDAC6, was not altered in FTLD-TDP and MSA relative to controls. These findings suggest that the induced level of HDAC6 in the brain is insufficient for manifestation of its activity in FTLD-TDP and MSA.     

A novel TK-NOG based humanized mouse model for the study of HBV and HCV infections

The immunodeficient mice transplanted with human hepatocytes are available for the study of the human hepatitis viruses. Recently, human hepatocytes were also successfully transplanted in herpes simplex virus type-1 thymidine kinase (TK)-NOG mice. In this study, we attempted to infect hepatitis virus in humanized TK-NOG mice and urokinase-type plasminogen activator-severe combined immunodeficiency (uPA–SCID) mice. TK-NOG mice were injected intraperitoneally with 6 mg/kg of ganciclovir (GCV), and transplanted with human hepatocytes. Humanized TK-NOG mice and uPA/SCID mice were injected with hepatitis B virus (HBV)- or hepatitis C virus (HCV)-positive human serum samples. Human hepatocyte repopulation index (RI) estimated from human serum albumin levels in TK-NOG mice correlated well with pre-transplantation serum ALT levels induced by ganciclovir treatment. All humanized TK-NOG and uPA–SCID mice injected with HBV infected serum developed viremia irrespective of lower replacement index. In contrast, establishment of HCV viremia was significantly more frequent in TK-NOG mice with low human hepatocyte RI (<70%) than uPA–SCID mice with similar RI. Frequency of mice spontaneously in early stage of viral infection experiment (8 weeks after injection) was similar in both TK-NOG mice and uPA–SCID mice. Effects of drug treatment with entecavir or interferon were similar in both mouse models. TK-NOG mice thus useful for study of hepatitis virus virology and evaluation of anti-viral drugs.     

Calcium-Dependent Interaction Sites of Tropomyosin on Reconstituted Muscle Thin Filaments with Bound Myosin Heads as Studied by Site-Directed Spin-Labeling

To identify the interaction sites of Tm, we measured the rotational motion of a spin-label covalently bound to the side chain of a cysteine that was genetically incorporated into rabbit skeletal muscle tropomyosin (Tm) at positions 13, 36, 146, 160, 174, 190, 209, 230, 271, or 279. Most of the Tm residues were immobilized on actin filaments with myosin-S1 bound to them. The residues in the mid-portion of Tm, namely, 146, 174, 190, 209, and 230, were mobilized when the troponin (Tn) complex bound to the actin-Tm-S1 filaments. The addition of Ca²⁺ ions partially reversed the Tn-induced mobilization. In contrast, residues at the joint region of Tm, 13, 36, 271, and 279 were unchanged or oppositely changed. All of these changes were detected using a maleimide spin label and less obviously using a methanesulfonate label. These results indicated that Tm was fixed on thin filaments with myosin bound to them, although a small change in the flexibility of the side chains of Tm residues, presumably interfaced with Tn, actin and myosin, was induced by the binding of Tn and Ca²⁺. These findings suggest that even in the myosin-bound (open) state, Ca²⁺ may regulate actomyosin contractile properties via Tm.     

Elucidations of the Catalytic Cycle of NADH-Cytochrome b5 Reductase by X-ray Crystallography: New Insights into Regulation of Efficient Electron Transfer

NADH-Cytochrome b₅ reductase (b5R), a flavoprotein consisting of NADH and flavin adenine dinucleotide (FAD) binding domains, catalyzes electron transfer from the two-electron carrier NADH to the one-electron carrier cytochrome b₅ (Cb5). The crystal structures of both the fully reduced form and the oxidized form of porcine liver b5R were determined. In the reduced b5R structure determined at 1.68 Å resolution, the relative configuration of the two domains was slightly shifted in comparison with that of the oxidized form. This shift resulted in an increase in the solvent-accessible surface area of FAD and created a new hydrogen-bonding interaction between the N5 atom of the isoalloxazine ring of FAD and the hydroxyl oxygen atom of Thr66, which is considered to be a key residue in the release of a proton from the N5 atom. The isoalloxazine ring of FAD in the reduced form is flat as in the oxidized form and stacked together with the nicotinamide ring of NAD⁺. Determination of the oxidized b5R structure, including the hydrogen atoms, determined at 0.78 Å resolution revealed the details of a hydrogen-bonding network from the N5 atom of FAD to His49 via Thr66. Both of the reduced and oxidized b5R structures explain how backflow in this catalytic cycle is prevented and the transfer of electrons to one-electron acceptors such as Cb5 is accelerated. Furthermore, crystallographic analysis by the cryo-trapping method suggests that re-oxidation follows a two-step mechanism. These results provide structural insights into the catalytic cycle of b5R.     

An anti-sulfatide antibody O4 immunoprecipitates sulfatide rafts including Fyn, Lyn and the G protein α subunit in rat primary immature oligodendrocytes

The association of sulfatide with specific proteins in oligodendrocytes was examined by co-immunoprecipitation with an anti-sulfatide antibody. Protein kinase activity was detected in precipitates with a monoclonal antibody to sulfatide (O4) from the rat primary immature oligodendrocytes. We conducted in vitro kinase assay of tyrosine phosphorylated proteins of 80, 59, 56, 53 and 40 kDa by gel electrophoresis. Of these proteins, the proteins of 59 kDa and 53/56 kDa were identified as the Src family tyrosine kinases Fyn and Lyn on the basis of their sequential immunoprecipitation with anti-Fyn and anti-Lyn antibodies, respectively. The 40 kDa protein was identified as the α subunit of the heterotrimeric G protein. These observations suggest that O4 immunoprecipitates sulfatide rafts including Fyn, Lyn and the α subunit of the heterotrimeric G protein.     

Evaluation of antioxidant activity of leafy vegetables and beans with myoglobin method

Key message

Antioxidant activity of seven leafy vegetables and four beans against five reactive oxygen species and reactive nitrogen species was clearly characterized with a protocol using myoglobin as a reporter probe.

Abstract

Antioxidant activity of seven leafy vegetables and four beans against peroxyl radical, hydroxyl radical, hypochlorite ion, and peroxynitrite ion has been measured using myoglobin as a reporter probe (myoglobin method). Conventional DPPH method was also used to evaluate antioxidant activity of the samples. Difference of activity against different reactive oxygen species (ROS) and reactive nitrogen species (RNS) was characterized by plotting the data in a 5-axe cobweb chart. This plot clearly showed the characteristics of the antioxidant activity of the leafy vegetables and the beans. The samples examined in this work were categorized into four groups. (1) The samples showed high antioxidant activity against all ROS and RNS: daikon sprout, spinach, Qing-geng-cai, and onion. (2) The samples showed high antioxidant activity against peroxyl radical: red bean and soy bean. (3) The samples showed high antioxidant against hypochlorite ion: broccoli floret, cabbage, and Chinese cabbage. (4) The samples showed weak antioxidant activity against all ROS and RNS: cowpea and common beans. Our protocol is probably useful to characterize antioxidant activity of the crops of different cultivars, the crops obtained in different growing environments and growing seasons, the crops harvested at different age, and the crops stored in the different conditions, as well as the changes of activity during cooking process of the crops.     

Extracellular histone induces plasma hyaluronan-binding protein (factor VII activating protease) activation in vivo

Plasma hyaluronan-binding protein (PHBP), an activator of factor VII and prourokinase, is a serine protease circulating as a single-chain proenzyme (pro-PHBP). Pro-PHBP converts to the active two-chain form through autoproteolysis, and effectors that modulate autoactivation can regulate PHBP-mediated processes. Here, we show that histone promotes pro-PHBP autoactivation in vivo. Histone bound to pro-PHBP and promoted intermolecular pro-PHBP binding. Histone-mediated pro-PHBP activation in plasma leads to the formations of bradykinin and PHBP−α₂-antiplasmin complex as well as histone degradation. Pro-PHBP activation was observed in the circulation of mice after injection of histone or lipopolysaccharide, which induced septic response accompanying extracellular histone release. Our results suggest pathophysiological relevance of histone-dependent pro-PHBP activation in hyperinflammatory process.