An inducible lactone hydrolase yielding 2,5-diphenyl-3-hydroxy-4-oxo-2-hexendioic acid from pulvinic acid

The metabolism of vulpinic acid by an unclassified soil micro-organism was studied. A new compound, 2,5-diphenyl-3-hydroxy-4-oxo-2-hexendioic acid (DHOHA) was isolated from the reaction mixture of a cell-free preparation and pulvinic acid. The existence of a hydrolase which catalyses the conversion of vulpinic acid to pulvinic acid was detected in cell-free preparation, and an inducible lactone hydrolase capable of converting pulvinic acid to DHOHA was purified 130-fold and characterized. This enzyme had a MW of ca 34 000, a K_m for pulvinic acid at pH optimum (pH 7.0) less than 10 ^{? 6} M, pI = 5.0, and was inhibited by p-chloromercuriphenylsulfonate and diethylpyrocarbonate. The enzyme was highly specific for pulvinic acid. The initial degradative steps proposed for this organism are vulpinic acid → pulvinic acid → DHOHA.     

Secretory expression of a glutamic-acid-specific endopeptidase (SPase) from Staphylococcus aureus ATCC12600 in Bacillus subtilis

Summary In order to obtain a large quantity of glutamic-acid-specific endopeptidase of Staphylococcus aureus ATCC12600 (SPase) without cultivating its pathogenic host bacterium, expression plasmids enabling secretion of SPase from Bacillus subtilis were constructed by inserting the SPase gene into B. subtilis-Escherichia coli shuttle vectors. B. subtilis harbouring a simple recombinant plasmid containing the coding and the 5-flanking regions of SPase in the shuttle vector pHY300PLK secreted 22 mg/l of SPase into the medium. As this level was lower than that of the natural strain (45 mg/l), we tried to increase the expression level by constructing a series of hybrid plasmids with the following features: (1) the terminator sequence of the alkaline protease gene from B. subtilis, (2) the promoter and the leader sequences of the -amylase gene or of alkaline protease gene from B. amyloliquefaciens, (3) the vector pHY300PLK and the fused vector of pHY300PLK and pUB110. By using a variety of hybrid plasmids, the resulting transformants secreted SPase at levels of 33–120 mg/l. The recombinant SPase isolated from the medium was indistinguishable from the natural one with respect to its behaviour on sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting as well as its enzyme activity.Correspondence to: S. Kakudo     

Impairment of Protein Synthesis in the Retinal Tissue in Diabetic Rabbits: Secondary Reduction of Fast Axonal Transport

Protein biosynthesis in the retina and fast axonal transport along the optic pathway were studied in rabbits in which diabetes had been experimentally induced. Retinal protein biosynthesis and axonal transport were significantly reduced in the diabetic rabbits, and the reduction was correlated to the severity of the diabetes. The "somal delay time' was only slightly elongated and the O/R ratio was fairly constant in the various levels of blood glucose; thus intrasomal protein movement seems to be less affected in diabetic rabbits. Velocity and the distribution pattern of axonally transported protein remained unaffected in the diabetic rabbits. These findings suggest that a disturbance in the metabolism in the cell body is the most important factor related to quantitative reduction of fast axonal transport in diabetic rabbits.     

Optical Activity Governed by Local Chiral Structures in Two‐Dimensional Curved Metallic Nanostructures

Chiral nanostructures show macroscopic optical activity. Local optical activity and its handedness are not uniform in the nanostructure, and are spatially distributed depending on the shape of the nanostructure. In this study we fabricated curved chain nanostructures made of gold by connecting linearly two or more arc structures in a two‐dimensional plane. Spatial features of local optical activity in the chain structures were evaluated with near‐field circular dichroism (CD) imaging, and analyzed with the aid of classical electromagnetic simulation. The electromagnetic simulation predicted that local optical activity appears at inflection points where arc structures are connected. The handedness of the local optical activity was dependent on the handedness of the local chirality at the inflection point. Chiral chain structures have odd inflection points and the local optical activity distributed symmetrically with respect to structural centers. In contrast, achiral chain structures have even inflection points and showed antisymmetric distribution. In the near‐field CD images of fabricated chain nanostructures, the symmetric and antisymmetric distributions of local CD were observed for chiral and achiral chain structures, respectively, consistent with the simulated results. The handedness of the local optical activity was found to be determined by the handedness of the inflection point, for the fabricated chain structures having two or more inflection points. The local optical activity was thus governed primarily by the local chirality of the inflection points for the gold chain structures. The total effect of all the inflection points in the chain structure is considered to be a predominant factor that determines the macroscopic optical activity. Chirality 28:540–544, 2016. © 2016 Wiley Periodicals, Inc.     

Characterization of a new phosphonocerebroside, N-methyl-2-aminoethylphosphonylglucosylceramide, from the antarctic krill, Euphausia superba

A novel phosphonglycosphingolipid was purified from the whole tissue of the antarctic krill, Euphausia superba by successive column chromatography on DEAE- and QAE-Sephadex and silicic acid (Iatrobeads). The structure was elucidated by means of IR, FAB-MS, 1H-NMR, GC and GC-MS analyses of the water-soluble products after complete and partial acid hydrolysis, and methylation analysis of a product of hydrogen fluoride degradation; it was identified to be a phosphonocerebroside, 6'-O-(N-methyl-2-aminoethylphosphonyl)Glcp beta 1----1ceramide. The ceramide moiety was composed of tetradecasphingenine and octadecasphingatriene as the main sphingoids, and monounsaturated C22- and C24-acids and their 2-hydroxy homologues as the major fatty acids.     

Effects of a novel gaseous antioxidative system containing a rosemary extract on the oxidation induced by nitrogen dioxide and ultraviolet radiation

Rosemary is commonly used as a spice and a flavoring agent in food processing. Although the antioxidative properties of its extracts have been investigated, there have been few reports on the volatile components of rosemary. We designed a novel antioxidative system which can generate the volatile constituents in the gaseous phase from a rosemary extract and evaluated the gaseous antioxidative activities against both lipid peroxidation and cell death induced by nitrogen dioxide and ultraviolet radiation. The antioxidative effects of the major volatile components on the oxidation of linoleic acid induced by azo compounds were also investigated in a solution. The volatile components in the novel antioxidative system suppressed the Jurkat cell death induced by nitrogen dioxide and the intracellular formation of reactive oxygen species in fibroblast cells induced by ultraviolet radiation. 1,8-Cineole among the volatile components exerted an antioxidative effect against the oxidation of linoleic acid in a solution induced by azo compounds and ultraviolet radiation. These data suggest that the volatile constituents of a rosemary extract had antioxidative properties and that gaseous exposure antioxidant is a promising method for promoting health.     

Detection of lipid peroxidation in vivo: total hydroxyoctadecadienoic acid and 7-hydroxycholesterol as oxidative stress marker

It is important to assess the oxidative injury in vivo accurately and inclusively, as the oxidative stress induced by various oxidants in a random and destructive fashion is considered to play an important role in the pathophysiology of a number of human disorders and diseases. We have developed an improved method for the measurement of lipid peroxidation in vivo, where total hydroxyoctadecadienoic acids (HODE) and 7-hydroxycholesterol (FCOH) were determined by GC/MS analysis from physiological samples after reduction with sodium borohydride and saponification by potassium hydroxide. In this method, both free and ester forms of hydroperoxides and ketones as well as hydroxides of linoleate and cholesterol are measured as HODE and FCOH, respectively. The ratio of stereo-isomers, (E,E)-HODE/(E,Z)-HODE, could be also measured. The plasma concentrations of total HODE were obtained as 76.5, 666 and 2225 nM for human, rat and mouse, respectively. It was found that HODE and FCOH could be measured satisfactorily by the present method from plasma, erythrocyte and urine of humans and experimental animals. It was also found that HODE in urine arose from both free and ester forms, while 8-iso-prostaglandin F2alpha was present primarily as a free acid form. As the concentrations of HODE were much higher than 8-iso-prostaglandin F2alpha, HODE may well be used as a good oxidative marker in vivo.     

Dual effects of acupuncture on gastric motility in conscious rats

The effects of manual acupuncture on gastric motility were investigated in 35 conscious rats implanted with a strain gauge transducer. Twenty (57.1%) rats showed no cyclic groupings of strong contractions (type A), whereas 15 (42.9%) rats showed the phase III-like contractions of the migrating motor complex (type B) in the fasting gastric motility. Acupuncture at the stomach (ST)-36 (Zusanli), but not on the back [Weishu, bladder (BL)-21], increased the peak amplitude of contractions to 172.4 +/- 25.6% of basal in the type A rats (n = 20, P < 0.05). On the other hand, the motility index for 60 min after the acupuncture was not affected by the acupuncture in this group. On the contrary, acupuncture decreased the peak amplitude and motility index to 72.9 +/- 14.0% and 73.6 +/- 16.2% in the type B rats (n = 15, P < 0.05), respectively. The stimulatory and inhibitory effects of acupuncture observed in each type were reproducible on the separate days. In 70% of type A rats, acupuncture induced strong phase III-like contractions lasting for over 3 h that were abolished by atropine, hexamethonium, atropine methyl bromide, and vagotomy. Naloxone significantly shortened the duration of the stimulatory effects from 3.52 +/- 0.21 to 1.02 +/- 0.15 h (n = 3, P < 0.05). These results suggest that acupuncture at ST-36 induces dual effects, either stimulatory or inhibitory, on gastric motility. The stimulatory effects are mediated in part via vagal efferent and opioid pathways.     

Comparative study on the action of tocopherols and tocotrienols as antioxidant: chemical and physical effects

alpha-Tocopherol is known as the most abundant and active form of vitamin E homologues in vivo, but recently the role of other forms of vitamin E has received renewed attention. The antioxidant properties were compared for alpha-, beta-, gamma- and delta-tocopherols and tocotrienols. The following results were obtained: (1). the corresponding tocopherols and tocotrienols exerted the same reactivities toward radicals and the same antioxidant activities against lipid peroxidation in solution and liposomal membranes; (2). tocopherols gave more significant physical effect than tocotrienols on the increase in rigidity at the membrane interior; (3). tocopherols and tocotrienols showed similar mobilities within the membranes, but tocotrienols were more readily transferred between the membranes and incorporated into the membranes than tocopherols; (4). alpha-tocopherol and alpha-tocotrienol, but not the other forms, reduced Cu(II) to give Cu(I) together with alpha-tocopheryl and alpha-tocotrienyl quinones, respectively and exerted prooxidant effect in the oxidation of methyl linoleate in SDS micelles.