Cell Structure of Yeasts Grown Anaerobically in Aspergillus orpzae-Proteolipid-supplemented Media

Effects of Aspergillus oryzae-proteolipid (PL) on the formation of high concentrations of alcohol, more than 20% as in sake brewing, have been studied. Electron microscopy revealed that there were no vacuoles but many lipid deposits in cytoplasm of the alcoholtolerant cells of Saccharomyces sake Kyokai No. 7, which were obtained by the anaerobic culture supplemented with PL. Sphaeroplasts from the alcohol-tolerant cells were stable in 20% alcohol, whereas those from the untolerant cells grown anaerobically in the PL-unsupplemented media were ruptured. The cell membrane became alcohol-endurable in the anaerobic cultures with PL. Synthetic phospholipid promoted yeast growth and the fermentative activity, whereas a small amount of sterol ester enhanced the alcohoi-endurability. The supplementation of both lipids anaerobically induced physiological properties characteristic of the alcohol-tolerant cells.     

Isolation and Structure of Neoannonin,a Novel Insecticidal Compound from the Seeds of Annona squamosa

Two insecticidal compounds were isolated from the seeds of Annona squamosa. One was identified as annonin (squamocin) and the other was characterized as a novel dihydroxy-bistetrahydrofuran fatty acid lactone (acetogenin) with 35 carbons. These two compounds were toxic to eggs, larvae and adults of the fruitfly.     

The Chemistry of Ilexol. I.

Ilexol, a new triterpenoid alcohol isolated from the barks of several species of the Genus Ilex, has now been oxidized with chromium trioxide to yield a ketone, designated as ilexone of C₃₀H₄₈O, m.p. 239.5–240.5°, and – 68.74°, which gives oxirne of m. p. 256.5–257.5°, 2,4-dinitrophenylhydrazone of m.p. 260–261°, and positive Zimmermann test.

On reduction with sodium and ethanol as well as with lithium aluminum hydride, it has been shown that ilexone is regenerated to yield an alcohol proved to be identical with natural ilexol.

On reduction according to the procedure of Huang-Minion, ilexone has been shown to yield an oxygen-free compound, designated as ilexene of C₃₀H₅₀, and m.p. 241–242°.

It seems most likely that ilexol might be a triterpenoid alcohol of C₃₀H₄₉OH, an alcoholic hydroxyl group of which is secondary in nature, and moreover attached at C–3 in its molecule.     

The Heterogeneity of the 7S Soybean Protein by Sepharose Gel Chromatography and Disc Gel Electrophoresis

Two kinds of N-acetylmuramidase, M-1 and M-2 enzymes, that were isolated from the cultural broth of Stm. globisporus 1829, were remarkably different in amino acid composition, immunological properties and modes of lytic action from each other. The M-1 enzyme was composed of 186 amino acid residues of which two moles were of half cystine, while the M-2 enzyme was composed of 99 amino acid residues with no cysteine. The hydrolyzing action of the M-2 enzyme was suppressed by the presence of an O-acetyl group on muramic acid residues in the peptidoglycan moiety, while that of the M-l enzyme was independent of the presence of O-acetyl groups. However, the hydrolyzing activity of both enzymes was enhanced when some muramic acid residues were substituted with stem peptides containing alanine, isoglutamine and lysine.     

The Purification of Soybean 11S Globulin with ConA-Sepharose 4B and Sepharose 6B

Kinetics of the acyl transfer catalyzed by Xanthomonas α-amino acid ester hydrolase was studied. The enzyme hydrolyzed d-α-phenylglycine methyl ester (d-PG-OMe) to give equimolar amounts of d-α-phenylglycine and methanol. With d-PG-OMe as an acyl donor and 7-amino-3-deacetoxy-cephalosporanic acid (7-ADCA) as an acyl acceptor, the enzyme transferred the acyl group from d-PG-OMe to 7-ADCA in competition with water. The addition of amine nucleophiles (7-ADCA and 6-aminopenicillanic acid) decreased the molecular activity (k_o) of the enzyme-catalyzed hydrolysis of d-PG-OMe, whereas it did not alter the Michaelis constant (K_M), and plots of l/k_o against the initial concentration of a nucleophile (n_o) gave a straight line. These results support the assumptions that the overall process for hydrolysis and acyl transfer proceeds through a common acyl-enzyme intermediate, that the acylation step of the enzyme is rate-limiting, and that the transfer competes with the hydrolysis of the acyl donor.     

Axonal protection by Nmnat3 overexpression with involvement of autophagy in optic nerve degeneration

Axonal degeneration often leads to the death of neuronal cell bodies. Previous studies demonstrated the crucial role of nicotinamide mononucleotide adenylyltransferase (Nmnat) 1, 2, and 3 in axonal protection. In this study, Nmnat3 immunoreactivity was observed inside axons in the optic nerve. Overexpression of Nmnat3 exerts axonal protection against tumor necrosis factor-induced and intraocular pressure (IOP) elevation-induced optic nerve degeneration. Immunoblot analysis showed that both p62 and microtubule-associated protein light chain 3 (LC3)-II were upregulated in the optic nerve after IOP elevation. Nmnat3 transfection decreased p62 and increased LC3-II in the optic nerve both with and without experimental glaucoma. Electron microscopy showed the existence of autophagic vacuoles in optic nerve axons in the glaucoma, glaucoma+Nmnat3 transfection, and glaucoma+rapamycin groups, although preserved myelin and microtubule structures were noted in the glaucoma+Nmnat3 transfection and glaucoma+rapamycin groups. The axonal-protective effect of Nmnat3 was inhibited by 3-methyladenine, whereas rapamycin exerted axonal protection after IOP elevation. We found that p62 was present in the mitochondria and confirmed substantial colocalization of mitochondrial Nmnat3 and p62 in starved retinal ganglion cell (RGC)-5 cells. Nmnat3 transfection decreased p62 and increased autophagic flux in RGC-5 cells. These results suggest that the axonal-protective effect of Nmnat3 may be involved in autophagy machinery, and that modulation of Nmnat3 and autophagy may lead to potential strategies against degenerative optic nerve disease.     

An Archaeal Homolog of Proteasome Assembly Factor Functions as a Proteasome Activator

Assembly of the eukaryotic 20S proteasome is an ordered process involving several proteins operating as proteasome assembly factors including PAC1-PAC2 but archaeal 20S proteasome subunits can spontaneously assemble into an active cylindrical architecture. Recent bioinformatic analysis identified archaeal PAC1-PAC2 homologs PbaA and PbaB. However, it remains unclear whether such assembly factor-like proteins play an indispensable role in orchestration of proteasome subunits in archaea. We revealed that PbaB forms a homotetramer and exerts a dual function as an ATP-independent proteasome activator and a molecular chaperone through its tentacle-like C-terminal segments. Our findings provide insights into molecular evolution relationships between proteasome activators and assembly factors.     

Functional and Structural Analysis of a β-Glucosidase Involved in β-1,2-Glucan Metabolism in Listeria innocua

Despite the presence of β-1,2-glucan in nature, few β-1,2-glucan degrading enzymes have been reported to date. Recently, the Lin1839 protein from Listeria innocua was identified as a 1,2-β-oligoglucan phosphorylase. Since the adjacent lin1840 gene in the gene cluster encodes a putative glycoside hydrolase family 3 β-glucosidase, we hypothesized that Lin1840 is also involved in β-1,2-glucan dissimilation. Here we report the functional and structural analysis of Lin1840. A recombinant Lin1840 protein (Lin1840r) showed the highest hydrolytic activity toward sophorose (Glc-β-1,2-Glc) among β-1,2-glucooligosaccharides, suggesting that Lin1840 is a β-glucosidase involved in sophorose degradation. The enzyme also rapidly hydrolyzed laminaribiose (β-1,3), but not cellobiose (β-1,4) or gentiobiose (β-1,6) among β-linked gluco-disaccharides. We determined the crystal structures of Lin1840r in complexes with sophorose and laminaribiose as productive binding forms. In these structures, Arg572 forms many hydrogen bonds with sophorose and laminaribiose at subsite +1, which seems to be a key factor for substrate selectivity. The opposite side of subsite +1 from Arg572 is connected to a large empty space appearing to be subsite +2 for the binding of sophorotriose (Glc-β-1,2-Glc-β-1,2-Glc) in spite of the higher K_m value for sophorotriose than that for sophorose. The conformations of sophorose and laminaribiose are almost the same on the Arg572 side but differ on the subsite +2 side that provides no interaction with a substrate. Therefore, Lin1840r is unable to distinguish between sophorose and laminaribiose as substrates. These results provide the first mechanistic insights into β-1,2-glucooligosaccharide recognition by β-glucosidase.