Peroxyoxalate chemiluminescent assay for oxidase activities based on detecting enzymatically formed hydrogen peroxide

A sensitive peroxyoxalate chemiluminescent (PO-CL) assay for activities of oxidases (uricase, choline oxidase, cholesterol oxidase and xanthine oxidase) which catalyse a formation of hydrogen peroxide was developed using 4,4′-oxalyl-bis[(trifluoromethylsulphonyl)imino]trimethylene-bis(4-methylmorpholinium)trifluoromethanesulphonate as a chemiluminogenic reagent and 2,4,6,8-tetramorpholinopyrimido[5,4-d]pyrimidine as a fluorophore. The standard curve for hydrogen peroxide was linear over the range 1 × 10^?7-1 × 10^?4 mol/L. Relative standard deviations for oxidase assays were 5.1–12.7% (n = 10). Detection limits were 1 × 10^?3 U/mL for uricase, 5 × 10^?4 U/mL for choline oxidase, 5 × 10^?3 U/mL for cholesterol oxidase and 5 × 10^?4 U/mL xanthine oxidase (sample to blank ratio, 3).     

Annual survival rate and age structure of habu,Trimeresurus flavoviridis (Viperidae), on Minna island,Japan: Estimation from removal trapping

A population of a viperid snake,Trimeresurus flavoviridis, was studied over 10 years by removal trapping on a small subtropical island in Japan. The sex ratio of trapped individuals changed seasonally, but was not biased to either sex in the whole sample of 258 individuals. The age of each individual was estimated through the size structure and the age-size relationship. The minimum number of individuals at the beginning of the study was estimated through accumulating older individuals trapped in the subsequent years. By assuming an annual natural survival rate in the course of this accumulation, an age structure was simulated which led to calculate a resulted natural survival rate. The assumed rate of 0.62 fitted best to the resulted one. The annual trapped proportion estimated on the simulated absolute number of individuals was higher in older individuals than in younger ones with the overall mean of 16%.     

Presynaptic α2 Adrenoceptors Inhibit Glutamate Release from Rat Spinal Cord Synaptosomes

Abstract: The presynaptic regulation of amino acid release from nerve terminals was investigated using synaptosomes prepared from the rat spinal cord. The basal releases of endogenous glutamate (Glu), aspartate (Asp), and γ-amino-butyric acid (GABA) were 34.6, 21.5, and 10.0 pmol/min/mg of protein, respectively. Exposure to a depolarizing concentration of KCl (30 m M ) evoked 2.7-, 1.5-, and 2.9-fold increases in Glu, Asp, and GABA release, respectively. Clonidine reduced the K⁺-evoked overflow of Glu to 56% of the control overflow with a potency (IC₅₀) of 17 n M , but it did not affect K⁺-evoked overflow of Asp, GABA, and their basal releases. Similarly, noradrenaline inhibited the K⁺-evoked overflow of Glu, although phenylephrine and isoproterenol showed no effect. The inhibitory effect of clonidine was counteracted by α₂-adrenoceptor antagonists, rauwolscine, yohimbine, and idazoxan, regardless of the imidazoline structures. Because Glu is considered a neurotransmitter of primary afferents that transmit both nociceptive and nonnociceptive stimuli in the spinal cord, these data suggest that part of Glu release may be regulated by the noradrenergic system through α₂ adrenoceptors localized on the primary afferent terminals.     

W-heterochromatin of chicken; its unusual DNA components,late replication,and chromatin structure

About 65% of DNA in the chicken W chromosome has been shown to consist ofXhoI andEcoRI family repetitive sequences. These sequences showed remarkable delay in the electrophoretic mobility at low temperature on a polyacrylamide gel. Three dimensional structures of the 0.7-kbXhoI and the 1.2-kbEcoRI family repeating units were estimated to be irregular solenoids using a computer program based on wedge angles of all the 16 dinucleotide steps. Fluorescencein situ hybridization demonstrated that these two family sequences were localized in a major heterochromatic body in an interphase nucleus. Incorporation of bromodeoxyuridine into the W chromosome in the synchronous culture of MSB-1 cells occurred about 1 h later than the peak of S phase. The chromatin structure formed alongXhoI andEcoRI family sequences was suggested to be different from the total chromatin or chromatin containing the β-actin gene sequence in that the linker DNA lengths of the former were significantly longer. Fractionation of theHaeIII-digested MSB-1 nuclei yielded a chromatin fraction in whichXhoI family sequences were partially enriched. Several DNA-binding proteins showing higher affinity for theXhoI family sequence were present in this fraction.     

Improved strain distribution patterns of SMXA recombinant inbred strains by microsatellite markers.

To develop SMXA recombinant inbred (RI) strains as more valuable genetic resources, 302 microsatellite (Mit) loci were added to the strain distribution patterns (SDP) reported previously. The improved SDP were constructed in a total of 1085 loci containing 484 Mit markers, 571 restriction landmark genomic scanning (RLGS) spot markers and 30 others. This substantially improved SDP can be freely accessed on our homepage (http://www.med.nagoya-u.ac.jp/sisetu/SDP.htm).     

Effects of detergents on the secondary structures of prion protein peptides as studied by CD spectroscopy.

Pathogenic prion proteins (PrP(Sc)) are thought to be produced by alpha-helical to beta-sheet conformational changes in the normal cellular prion proteins (PrP(C)) located solely in the caveolar compartments. In order to inquire into the possible conformational changes due to the influences of hydrophobic environments within caveolae, the secondary structures of prion protein peptides were studied in various kinds of detergents by CD spectra. The peptides studied were PrP(129-154) and PrP(192-213); the former is supposed to assume beta-sheets and the latter alpha-helices, in PrP(Sc). The secondary structure analyses for the CD spectra revealed that in buffer solutions, both PrP(129-154) and PrP(192-213) mainly adopted random-coils (approximately 60%), followed by beta-sheets (30%-40%). PrP(129-154) showed no changes in the secondary structures even in various kinds of detergents such as octyl-beta-D-glucopyranoside (OG), octy-beta-D-maltopyranoside (OM). sodium dodecyl sulfate (SDS), Zwittergent 3-14 (ZW) and dodecylphosphocholine (DPC). In contrast, PrP(192-213) changed its secondary structure depending on the concentration of the detergents. SDS, ZW, OG and OM increased the alpha-helical content, and decreased the beta-sheet and random-coil contents. DPC also increased the alpha-helical content, but to a lesser extent than did SDS, ZW, OG or OM. These results indicate that PrP(129-154) has a propensity to adopt predominantly beta-sheets. On the other hand, PrP(192-213) has a rather fickle propensity and varies its secondary structure depending on the environmental conditions. It is considered that the hydrophobic environments provided by these detergents may mimic those provided by gangliosides in caveolae, the head groups of which consist of oligosaccharide chains containing sialic acids. It is concluded that PrP(C) could be converted into a nascent PrP(Sc) having a transient PrP(Sc) like structureunder the hydrophobic environments produced by gangliosides.     

Reversed-phase HPLC determination of chlorophyll a' and phylloquinone in Photosystem I of oxygenic photosynthetic organisms. Universal existence of one chlorophyll a' molecule in Photosystem I.

Chlorophyll (Chl) a', the C132-epimer of Chl a, is a constituent of the primary electron donor (P700) of Photosystem (PS) I of a thermophilic cyanobacterium Synechococcus (Thermosynechococcus) elongatus, as was recently demonstrated by X-ray crystallography. To determine whether PS I of oxygenic photosynthetic organisms universally contains one molecule of Chl a', pigment compositions of thylakoid membranes and PS I complexes isolated from the cyanobacteria T. elongatus and Synechocystis sp. PCC 6803, the green alga Chlamydomonas reinhardtii, and the green plant spinach, were examined by simultaneous detection of phylloquinone (the secondary electron acceptor of PS I) and Chl a' by reversed-phase HPLC. The results were compared with the Chl a/P700 ratio determined spectrophotometrically. The Chl a'/PS I ratios of thylakoid membranes and PS I were about 1 for all the organisms examined, and one Chl a' molecule was found in PS I even after most of the peripheral subunits were removed. Chl a' showed a characteristic extraction behaviour significantly different from the bulk Chl a in acetone/methanol extraction upon varying the mixing ratio. These findings confirm that a single Chl a' molecule in P700 is the universal feature of PS I of the Chl a-based oxygenic photosynthetic organisms.     

The Chromosomal Organization of the Human Endogenous Retrovirus-like Sequence HERV-H: Clustering of the HERV-H Sequences in a 300-kb Region Close to the GRPR Locus on the X Chromosome

Within the haploid genome there are approximately 1,000 copiesof the human endogenous retroviruslike sequence, HERV-H. Althoughthese sequences are scattered throughout the entire genome,in situ hybridization experiments revealed that there are discreteclusters positioned on chromosomes 1p and 7q. In this study,we have located three HERV-H sequences which were unexpectedlyclustered within a 300-kilobase region close to the GRPR locuson the X chromosome. In previous studies, no clusteringof thissequence has been reported at this locus. Our finding demonstratesthat, like other repetitive sequences, clustering of HERV-Hoccurs in the human genome, although these sequences may notalways be detected by in situ hybridization methods.     

Involvement of umuDCST genes in nitropyrene-induced -CG frameshift mutagenesis at the repetitive CG sequence in the hisD3052 allele of Salmonella typhimurium

Expression of the umuDC operon is required for UV and most chemical mutagenesis in Escherichia coli. The closely related species Salmonella typhimurium has two sets of umuDC-like operons, umuDC _ST on the chromosome and samAB on a 60-MDa cryptic plasmid. The roles of theumuDC-like operons in chemically induced frameshift mutagenesis of the hisD3052 allele of S. typhimurium were investigated. Introduction of a pBR322-derived plasmid carrying umuDCST increased the rate of reversion of hisD3052, following treatment with 1-nitropyrene (1-NP) or 1,8-dinitropyrene (1,-8DNP) tenfold and fivefold, respectively, whereas it did not substantially increase the rate of reversion induced by other frameshift mutagens, i.e. 2-nitrofluorene (2NF) and 2-amino- 3-methyldipyrido[1,2-a:3 ,2-d]imi-dazole (Glu-P-1). Introduction of a pBR322-derived plasmid carrying samAB did not increase the incidence of reversion of hisD3052 observed with any of the mutagens examined. Deletion of umuDC _STSubstantially lowered the reversion rate induced by l-NP or 1,8-DNP, but it did not affect reversion induced by 2-NF, Glu-P-1 or N-hydroxyacetylaminofluorene (N-OH-AAF). Deletion of samAB had little impact on reversion incidence induced by any of the five frameshift mutagens. DNA amplification using the polymerase chain reaction technique followed by restriction enzyme analysis using BssHII, suggested that the mutations induced by the five frameshift mutagens were all CG deletions at the CGCGCGCG sequence in hisD3052. These results suggest that umuDCST, but not samAB, is involved in the -2 frameshift mutagenesis induced by l-NP and 1,8-DNP at the repetitive CG sequence, whereas neither operon participates in induction of the same type of mutations by 2-NF, Glu-P-1 or N-OH-AAF.