Excitotoxicity-induced immediate surge in hippocampal prostanoid production has latent effects that promote chronic progressive neuronal death

Excitotoxicity is involved in neurodegenerative conditions. We investigated the pathological significance of a surge in prostaglandin production immediately after kainic acid (KA) administration [initial phase], followed by a sustained moderate elevation in prostaglandin level [late phase] in the hippocampus of juvenile rats. Numerous pyknotic hippocampal neurons were observed 72 h after KA treatment; this number remained elevated on days 10 and 30. Gross hippocampal atrophy was observed on days 10 and 30. Pre-treatment with indomethacin ameliorated neuronal death on days 10 and 30, and prevented hippocampal atrophy on day 30. Microglial response was moderated by the indomethacin pre-treatment. Blockade of only late-phase prostaglandin production by post-treatment with indomethacin ameliorated neuronal death on day 30. These findings suggest a role for initial-phase prostaglandin production in chronic progressive neuronal death, which is exacerbated by late-phase prostaglandin production. Blockade of prostaglandin production has therapeutic implications in preventing long-term neurological sequelae following excitotoxic brain damage.     

Biochemical Characterization of Lipoxygenase Lacking Mutants,L-l-less,L-2-less,and L-3-less Soybeans

In this paper, lipoxygenase lacking mutants were characterized in comparison with normal soybeans. The three lipoxygenase isozymes (L-l, L-2, and L-3) in crude seed extracts of normal soybeans were resolved clearly by an improved SDS-polyacrylamide gel electrophoresis. As expected, the three mutant types, L-l-less (P. I. 408251 and 133226), L-2-less (P. I. 86023), and L-3 less (Wasenatsu and Ichigowase) soybeans did not give L-l, L-2, and L-3 protein bands, respectively on a single dimension SDS gel.

An anti L-2 serum obtained from a rabbit reacted not only with the purified L-2 protein, but also partially with the purified L-l and L-3 proteins. By double immunodiffusion and immuno-disc gel electrofocusing analyses using the anti L-2 serum, L-l, L-2, and L-3 isozymes could not be detected in crude seed extracts from P.I. 408251, P. I. 86023, and Wasenatsu soybeans, respectively.

Three lipoxygenase activity peaks (L-l, L-2, and L-3 enzyme peaks) and a small unknown activity peak eluted right after the L-l peak were fractionated by DEAE-Sephacel column chromatography of crude seed extracts of Raiden (normal) soybeans. The chromatographic analyses have demonstrated that both the L-l and the unknown enzyme activities disappear completely in the L-l-less type soybean seeds, and that the L-2 and L-3 enzyme activities disappear completely in P. I. 86023 and the L-3-less type soybean seeds, respectively.     

Decarboxylation of Oxalic Acid during Ripening of Banana Fruit (Musa sapientum L.)

ABSTRACT

Japanese apricot, Prunus mume Sieb. et Zucc., biosynthesizes the l-phenylalanine-derived cyanogenic glucosides prunasin and amygdalin. Prunasin has biological properties such as anti-inflammation, but plant extraction and chemical synthesis are impractical. In this study, we identified and characterized UGT85A47 from Japanese apricot. Further, UGT85A47 was utilized for prunasin microbial production. Full-length cDNA encoding UGT85A47 was isolated from Japanese apricot after 5?- and 3?-RACE. Recombinant UGT85A47 stoichiometrically catalyzed UDP-glucose consumption and synthesis of prunasin and UDP from mandelonitrile. Escherichia coli C41(DE3) cells expressing UGT85A47 produced prunasin (0.64 g/L) from racemic mandelonitrile and glucose. In addition, co-expression of genes encoding UDP-glucose biosynthetic enzymes (phosphoglucomutase and UTP-glucose 1-phosphate uridiltransferase) and polyphosphate kinase clearly improved prunasin production up to 2.3 g/L. These results showed that our whole-cell biocatalytic system is significantly more efficient than the existing prunasin production systems, such as chemical synthesis.     

Red Pigment Formation by the Reaction of Oxidized Ascorbic Acid and Protein in a Food Model System of Low Moisture Content

Ascorbic acid-protein mixtures of low moisture content stored for 2~3 days in aerobic conditions at 60°C produced a red coloration, which was shown to have resulted from an amino-carbonyl reaction of oxidized ascorbic acid (dehydroascorbic acid, DHA) by the facts that DHA-casein or ovalbumin systems yielded a rapid red coloration in low moisture conditions as well as in an ethanol suspension. Zein showed only a weak red coloration with DHA. An apparent decrease in the free amino group, as determined by the TNBS method, was observed to be associated in parallel with the red pigment formation. The electrophoretic pattern of ovalbumin changed significantly upon incubation with DHA, together with the formation of the red pigment. The red pigment extracted from a DHA-casein system showed an absorption spectrum, TLC Rf value, and hydrolyzed products (DHA and scorbamic acid) identical to those of 2,2′-nitrilo di-2(2)-deoxy-l-ascorbic acid mono ammonium salt, produced from DHA and amino acid. Formation of the red pigment was also observed in the reaction of DHA with N_α-acetyl lysine. These results indicate that the ε-amino group of lysine in protein can be attributed to the formation of a red pigment identical with NDA.     

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.     

A Root Growth-promoting Factor,Capillarol, from Artemisia capillaris Thunb

A root growth-promoting factor was isolated from Artemisia capillaris Thunb. The chemical structure of this compound was elucidated as methyl 3-(3-methylbut-2-enoyl)-4-hydroxycinnamate (capillarol) on the basis of its spectroscopic analysis. At 5×10^–4m this compound promoted rice root growth to 180% of the control value.     

Secondary structural analysis of proteins based on 13C chemical shift assignments in unresolved solid-state NMR spectra enhanced by fragmented structure database

Magic-angle-spinning solid-state ¹³C NMR spectroscopy is useful for structural analysis of non-crystalline proteins. However, the signal assignments and structural analysis are often hampered by the signal overlaps primarily due to minor structural heterogeneities, especially for uniformly-¹³C,¹⁵N labeled samples. To overcome this problem, we present a method for assigning ¹³C chemical shifts and secondary structures from unresolved two-dimensional ¹³C–¹³C MAS NMR spectra by spectral fitting, named reconstruction of spectra using protein local structures (RESPLS). The spectral fitting was conducted using databases of protein fragmented structures related to ¹³C^α, ¹³C^β, and ¹³C′ chemical shifts and cross-peak intensities. The experimental ¹³C–¹³C inter- and intra-residue correlation spectra of uniformly isotope-labeled ubiquitin in the lyophilized state had a few broad peaks. The fitting analysis for these spectra provided sequence-specific C^α, C^β, and C′ chemical shifts with an accuracy of about 1.5 ppm, which enabled the assignment of the secondary structures with an accuracy of 79 %. The structural heterogeneity of the lyophilized ubiquitin is revealed from the results. Test of RESPLS analysis for simulated spectra of five different types of proteins indicated that the method allowed the secondary structure determination with accuracy of about 80 % for the 50–200 residue proteins. These results demonstrate that the RESPLS approach expands the applicability of the NMR to non-crystalline proteins exhibiting unresolved ¹³C NMR spectra, such as lyophilized proteins, amyloids, membrane proteins and proteins in living cells.     

Enhancement of Glossiness Perception by Retinal-Image Motion: Additional Effect of Head-Yoked Motion Parallax

It has been argued that when an observer moves, a contingent retinal-image motion of a stimulus would strengthen the perceived glossiness. This would be attributed to the veridical perception of three-dimensional structure by motion parallax. However, it has not been investigated whether the effect of motion parallax is more than that of retinal-image motion of the stimulus. Using a magnitude estimation method, we examine in this paper whether cross-modal coordination of the stimulus change and the observer''s motion (i.e., motion parallax) is essential or the retinal-image motion alone is sufficient for enhancing the perceived glossiness. Our data show that a retinal-image motion simulating motion parallax without head motion strengthened the perceived glossiness but that its effect was weaker than that of motion parallax with head motion. These results suggest the existence of an additional effect of the cross-modal coordination between vision and proprioception on glossiness perception. That is, motion parallax enhances the perception of glossiness, in addition to retinal-image motions of specular surfaces.     

Model studies for isolation of G-quadruplex-forming DNA sequences through a pull-down strategy with macrocyclic polyoxazole

G-quadruplexes (G4s) are non-B DNA structures present in guanine-rich regions of gene regulatory areas, promoters and CpG islands, but their occurrence and functions remain incompletely understood. Thus, methodology to identify G4 sequences is needed. Here, we describe the synthesis of a novel cyclic hepta-oxazole compound, L1Bio-7OTD (1), bearing a biotin affinity-tag as a tool to pull down G4 structures from mixtures of G4-forming and non G4-forming DNA sequences. We confirmed that it could pull down G4s associated with telomeres, bcl-2 gene, and c-kit gene.