SCA8 RAN polySer protein preferentially accumulates in white matter regions and is regulated by eIF3F

Spinocerebellar ataxia type 8 (SCA8) is caused by a bidirectionally transcribed CTG·CAG expansion that results in the in vivo accumulation of CUG RNA foci, an ATG‐initiated polyGln and a polyAla protein expressed by repeat‐associated non‐ATG (RAN) translation. Although RAN proteins have been reported in a growing number of diseases, the mechanisms and role of RAN translation in disease are poorly understood. We report a novel toxic SCA8 polySer protein which accumulates in white matter (WM) regions as aggregates that increase with age and disease severity. WM regions with polySer aggregates show demyelination and axonal degeneration in SCA8 human and mouse brains. Additionally, knockdown of the eukaryotic translation initiation factor eIF3F in cells reduces steady‐state levels of SCA8 polySer and other RAN proteins. Taken together, these data show polySer and WM abnormalities contribute to SCA8 and identify eIF3F as a novel modulator of RAN protein accumulation.     

Physicochemical characteristics and toxicity of nickel oxide particles calcined at different temperatures

The physicochemical characteristics and cytotoxicity of two types of commercial nickel oxide particles (black and green nickel oxide) and five types of nickel oxide particles prepared by calcination of the black nickel oxide at 600–1000°C were studied. Thermal analysis with mass spectroscopy showed that the black nickel oxide particles contained approximately 1.4% impurity, which seemed to be basic nickel carbonate. The calcination treatment at 600°C increased the nickel content and decreased the oxygen content, but these remained constant in the particles treated at higher temperatures (700–1000°C) and in the green nickel oxide particles. The water solubility of black nickel oxide particles was markedly greater than that of the other particles, especially in the first 24 h after mixing with water. The solubility of the calcined particles decreased with increasing calcination temperature. The cytotoxicity of these particles was evaluated by the viability of rat alveolar macrophages and by the inhibition of cell proliferation in Chinese hamster ovary cells. The black nickel oxide was the most cytotoxic of the particles examined, and this may be attributable, at least in part, to a rapid dissolution of nickel from the contained impurity. The toxicity of the calcined particles decreased with increasing calcination temperature. These results indicate that water solubility, which depends on calcination temperature, modulates the acute cytotoxicity of nickel oxide particles.     

Occurrence of the polyamines caldopentamine and homocaldopentamine in axenic cultures of the red tide flagellates Chattonella antiqua and Heterosigma akashiwo (Raphidophyceae)

The polyamines caldopentamine and homocaldopentamine were detected in axenic strains of Chattonella antiqua and Heterosigma akashiwo ( Raphidophyceae ), respectively, as well as spermidine, the most abundant polyamine in both phytoplankton species. Trace amounts of putrescine, diaminopropane and norspermine were also detected in both species. Spermine was detected only from C. antiqua . These long linear polyamines are characteristic components of thermophilic bacteria. The detection from two species of Raphidophyceae indicates that the occurrence of long linear polyamines is not restricted to thermophilic microorganisms.     

Non-Specific Association of Phytochrome to Nuclei during Isolation from Dark-Grown Pea (Pisum sativum cv. Alaska) Plumules.

Although phytochrome is known to regulate gene expression inplants, the location of the phytochrome molecules involved inthe response remains unclear. Thus the aim of this investigationwas to test the possibility that nuclei contain phytochrome.Nuclei were isolated from dark-grown pea (Pisum sativum cv.Alaska) plumules in the dark and the nuclear proteins were resolvedon SDS polyacrylamide gels and transferred to nitrocelluloseby western blotting. A significant amount of phytochrome wasdetected in the nuclear proteins by immunostaining using monoclonaland polyclonal anti-phytochrome antibodies. Most of the phytochromeassociated with the nuclei could not be removed by treatmentwith high salt and low magnesium, indicating that the bindingwas rather stable. However, the isolation of nuclei in the presenceof exogenously added [¹²⁵I]phytochrome of P_R form in the darksuggested that significant amount of the phytochrome detectedin the nuclei was derived from contamination by the solublefraction during isolation. It is an open question as to whether,besides this contaminated phytochrome, isolated nuclei endogenouslycontain very small amount of phytochrome. ⁴Present Address: Plant Molecular Biology and Biochemistry ResearchGroup, Departments of Biochemistry and Botany, University ofGlasgow, Glasgow G12 8QQ, U.K. (Received February 8, 1988; Accepted July 27, 1988)     

An assay method for cyclic AMP using high-performance liquid chromatography with pretreatment by alkaline phosphatase

A convenient method for determination of cyclic AMP is described. This nucleotide was determined using high-performance liquid chromatography after the treatment of tissue extracts by alkaline phosphatase. Tissue extracts contain large amounts of materials which interfere with the measurement of cyclic AMP on the chromatogram. By the alkaline phosphatase treatment, these materials were completely converted to compounds which no longer interfered with the measurement. This method enables the detection of 2 pmol cyclic AMP, and is applicable to various tissue extracts which contain at least 0.1 nmol cyclic AMP/g wet wt.     

Ultrastructural changes in differentiating neuroblastoma × glioma hybrid cells

The ultrastructure of differentiating neuroblastoma × glioma hybrid cells NG108-15 was observed. Cells cultured in growth medium showed undifferentiated features, while cells treated with dBcAMP became round and large, and extended thick long neurites. After 1 week in culture, cells showed features similar to those of normal neurons. The dense cored vesicles with diameters ranging from 60 to 170 nm were observed in differentiated NG108-15 cells, but clear vesicles were usually rare. However, in the case of co-culture with striated myotubes, clusters of clear vesicles appeared in the neurites and terminals. The timecourse of the differentiation process was correlated with results obtained by the electrophysiology and freeze-fracture.     

Mutations at the distal and proximal sites of cytochrome P-450d changed regio-specificity of acetanilide hydroxylations

Regio-specificities of acetanilide hydroxylations were studied for 11 distal, 9 proximal and 3 aromatic mutants of cytochrome P-450d. Ratios of turnover numbers among these products were remarkably changed depending on the mutants. For example, the ratio of turnover number, para:ortho:meta = 7:0.1:0.3 for the wild type changed to 11:4:3 for a distal mutant, Thr322Ala, or to 13:13:1 for a proximal mutant, Arg455Gly. It was suggested that regio-specificities of microsomal P-450 enzymes are controlled cooperatively by the whole structure of the protein molecules which influences the tertiary structure of the distal environment.     

Site-directed mutageneses of rat liver cytochrome P-450d: catalytic activities toward benzphetamine and 7-ethoxycoumarin

Catalytic activities toward benzphetamine and 7-ethoxycoumarin of 11 distal mutants, 9 proximal mutants, and 3 aromatic mutants of rat liver cytochrome P-450d were studied. A distal mutant Thr319Ala was not catalytically active toward benzphetamine, while this mutant retained activity toward 7-ethoxycoumarin. Distal mutants Gly316Glu, Thr319Ala, and Thr322Ala displayed higher activities (kcat/Km) toward 7-ethoxycoumarin that were 2.4-4.7-fold higher than that of the wild-type enzyme. Although kcat/Km values of four multiple distal mutants toward benzphetamine were less than half that of the wild type, activities of these mutants toward 7-ethoxycoumarin were almost the same as or higher than the wild-type activity toward this substrate. The distal double mutant Glu318Asp, Phe325Tyr showed 6-fold higher activity than the wild-type P-450d toward 7-ethoxycoumarin. Activities of the proximal mutants Lys453Glu and Arg455Gly toward both substrates were much lower (less than one-seventh) than the corresponding wild-type activities. Catalytic activities of three aromatic mutants, Phe425Leu, Pro427Leu, and Phe430Leu, toward benzphetamine were less than 7% of that of the wild type, while the activities of these aromatic mutants toward 7-ethoxycoumarin were more than 2.5 times higher than the wild-type activity toward this substrate. From these findings, in conjunction with a molecular model for P-450d, we suggest that (1) the relative importance to catalysis of various distal helix amino acids differs depending on the substrate and that these differences are associated with the size, shape, and flexibility of the substrate and (2) the proximal residue Lys453 appears to play a critical role in the catalytic activity of P-450d, perhaps by participating in forming an intermolecular electron-transfer complex.