Dorfin ubiquitylates mutant SOD1 and prevents mutant SOD1-mediated neurotoxicity

Amyotrophic lateral sclerosis (ALS) is a progressive paralytic disorder resulting from the degeneration of motor neurons in the cerebral cortex, brainstem, and spinal cord. The cytopathological hallmark in the remaining motor neurons of ALS is the presence of ubiquitylated inclusions consisting of insoluble protein aggregates. In this paper we report that Dorfin, a RING finger-type E3 ubiquitin ligase, is predominantly localized in the inclusion bodies of familial ALS with a copper/zinc superoxide dismutase (SOD1) mutation as well as sporadic ALS. Dorfin physically bound and ubiquitylated various SOD1 mutants derived from familial ALS patients and enhanced their degradation, but it had no effect on the stability of the wild-type SOD1. The overexpression of Dorfin protected against the toxic effects of mutant SOD1 on neural cells and reduced SOD1 inclusions. Our results indicate that Dorfin protects neurons by recognizing and then ubiquitylating mutant SOD1 proteins followed by targeting them for proteasomal degradation.     

Fatty acid composition of lipopolysaccharides of Vibrio cholerae 35A3 (Inaba), NIB 90 (Ogawa), and 4715 (Nag).

Considerable amounts of odd-numbered fatty acids, such as non-hydroxy C15 and C17 and 3-hydroxy C11 and C13 acids, were found in lipopolysaccharides from Vibrio cholerae 35A3 (Inaba).     

Nucleotide sequence of the thymidylate synthase B and dihydrofolate reductase genes contained in one Bacillus subtilis operon

The nucleotide sequence of the thymidylate synthase B (thyB) and dihydrofolate reductase (dfrA) gene regions from wild-type and trimethoprim-resistant (TpR) mutant strains of Bacillus subtilis 168 was determined. The sequenced region contains two open reading frames, ORF1 and ORF2, which correspond to thyB and dfrA, respectively, and overlap by one nucleotide. The thyB-dfrA genes encode 267 and 168 amino acid polypeptides, respectively, and are present in the order of thyB - dfrA in 5'----3' orientation. This gene order differs from those which have been found in other organisms so far. S1 mapping analysis indicated that both genes were transcribed from a single promoter located upstream from the thyB gene. Thus, the genes belong to an operon. A nucleotide substitution from 'A' in the wild type to 'C' in the TpR mutant was located in the dfrA gene region, with predicted conversion of isoleucine-95 (wild type) to leucine-95 (mutant) in dihydrofolate reductase (DHFR). It is suggested that the affinity between DHFR and Tp is reduced by this alteration.     

Identification of a factor that complementarily inhibits somatic embryogenesis with vanillyl benzyl ether in Japanese larch

In Japanese larch (Larix leptolepis Gordon), a well-developed suspensor forms during somatic embryogenesis. The suspensor is the essential tissue for development of the embryo proper. In high-cell-density culture, the embryogenic cells proliferate, but no somatic embryos form because suspensor development is suppressed. Previously, we identified vanillyl benzyl ether (VBE) as a novel factor suppressing suspensor development from the high-cell-density conditioned medium (HCM), but the inhibitory effect of VBE was weaker than that of HCM added. Therefore, this study attempted to identify another inhibitory factor in the culture medium. Induction of somatic embryos was performed in a medium containing both VBE and a fraction of each chromatogram extracted from the culture medium. Results of the bioassay showed that a fraction had strong inhibitory activity with VBE, but weak activity without it. By physicochemical analyses of the fraction, 4-[(phenylmethoxy)methyl]phenol was identified as an inhibitory factor of larch somatic embryogenesis.     

Pyrazolecarboxylic acid derivative induces systemic acquired resistance in tobacco

Systemic acquired resistance (SAR) is a potent innate immunity system in plants that is induced through asalicylic acid (SA)-mediated pathway. Here, we characterized 3-chloro-1-methyl-1H-pyrazole-5-carboxylic acid (CMPA) as an effective SAR inducer in tobacco. Soil drench application of CMPA induced PR gene expression and a broad range of disease resistance without antibacterial activity in tobacco. Both analysis of CMPA's effects on NahG transgenic tobacco plants and SA measurement in wild-type plants indicated that CMPA-induced resistance enhancement does not require SA. Therefore, it is suggested that CMPA induces SAR by triggering the signaling at the same level as or downstream of SA accumulation as do both benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester and N-cyanomethyl-2-chloroisonicotinamide.     

Stability of ribonuclease T2 from Aspergillus oryzae.

The stability of ribonuclease T2 (RNase T2) from Aspergillus oryzae against guanidine hydrochloride and heat was studied by using CD and fluorescence. RNase T2 unfolded and refolded reversibly concomitant with activity, but the unfolding and refolding rates were very slow (order of hours). The free energy change for unfolding of RNase T2 in water was estimated to be 5.3 kcal.mol-1 at 25 degrees C by linear extrapolation method. From the thermal unfolding experiment in 20 mM sodium phosphate buffer at pH 7.5, the Tm and the enthalpy change of RNase T2 were found to be 55.3 degrees C and 119.1 kcal.mol-1, respectively. From these equilibrium and kinetic studies, it was found that the stability of RNAse T2 in the native state is predominantly due to the slow rate of unfolding.     

Conformation of an Shc-derived phosphotyrosine-containing peptide complexed with the Grb2 SH2 domain

We have determined the structure of an Shc-derivedphosphotyrosine-containing peptide complexed with Grb2 SH2 based on intra-and intermolecular NOE correlations observed by a series of isotope-filteredNMR experiments using a PFG z-filter. In contrast to an extendedconformation of phosphotyrosine-containing peptides bound to Src, Syp andPLC SH2s, the Shc-derived peptide formed a turn at the +1 and +2positions next to the phosphotyrosine residue. Trp¹²¹, locatedat the EF1 site of Grb2 SH2, blocked the peptide binding in an extendedconformation. The present study confirms that eachphosphotyrosine-containing peptide binds to the cognate SH2 with a specificconformation, which gives the structural basis for the binding specificitybetween SH2s and target proteins.