Proton NMR resonance assignments and surface accessibility of tryptophan residues of a dimeric phospholipase A2 from Trimeresurus flavoviridis

Proton NMR spectra of a dimeric phospholipase A₂ from Trimeresurus flavoviridis have been recorded. N-1 proton resonances of the tryptophan indole rings have been detected and assigned to specific positions, Trp-3/Trp-30, Trp-68 and Trp-108, by comparing the spectra of the enzyme derivatives with tryptophans oxidized to differing extents. Photo-CIDNP experiments have revealed that Trp-68 and Trp-108 are exposed while Trp-3 and Trp-30 are buried in the molecule. This is consistent with the X-ray crystal structure of a homologous phospholipase A₂ from Crotalus atrox where residues 3 and 30 are located at a dimer interface, but inconsistent with the results of stepwise oxidation of tryptophan residues.     

Immunological and genomic analyses of two serotypes of avian paramyxovirus isolated from wild ducks in Japan

A total of 11 avian paramyxoviruses isolated from migrating feral ducks in Niigata, Japan, were characterized by serological and genomic analyses. Hemagglutination inhibition and immuno-double-diffusion tests with antisera specific for the isolated hemagglutinin-neuraminidase polypeptides of reference strains indicated that, of these, eight isolates possessed hemagglutinin-neuraminidase antigen closely related to that of duck/Hong Kong/D3/75, and the remaining three isolates possessed antigen closely related to that of duck/Hong Kong/199/77. RNA analysis of the eight isolates identified serologically as duck/Hong Kong/D3/75 by oligonucleotide mapping revealed that these isolates were genetically very similar to each other but different from the reference strain and isolates reported previously. The oligonucleotide maps of duck/Hong Kong/199/77-like isolates appeared to be very similar to each other, suggesting the same origin, but not to the duck/Hong Kong/199/77 virus.     

Free radical scavenging activity of novel thiazolidine‐2,4‐dione derivatives

Free radical activity towards superoxide anion radical (), hydroxyl radical (HO^?) and 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH^?) of a series of novel thiazolidine‐2,4‐dione derivatives (TSs) was examined using chemiluminescence, electron paramagnetic resonance (EPR) and EPR spin trapping techniques. 5,5‐Dimethyl‐1‐pyrroline‐N‐oxide (DMPO) was applied as the spin trap. Superoxide radical was produced in the potassium superoxide/18‐crown‐6 ether dissolved in dimethyl sulfoxide. Hydroxyl radical was generated in the Fenton reaction (Fe(II) + H₂O₂. It was found that TSs showed a slight scavenging effect (15–38% reduction at 2.5 mmol/L concentration) of the DPPH radical and a high scavenging effect of (41–88%). The tested compounds showed inhibition of HO^? ‐dependent DMPO‐OH spin adduct formation (the amplitude of EPR signal decrease ranged from 20 to 76% at 2.5 mmol/L concentration. Our findings present new group compounds of relatively high reactivity towards free radicals. Copyright © 2012 John Wiley & Sons, Ltd.     

Possible Involvement of Interleukin-1 in Cyclooxygenase-2Induction After Spinal Cord Injury in Rats

Abstract : A standardized compression injury of rat spinal cord brought about a time-dependent biphasic production of thromboxane A₂ (detected as thromboxane B₂) and prostaglandin I₂ (detected as 6-ketoprostaglandin F_1α. Thromboxane B₂ was predominant during the first 1 h, whereas the 6-ketoprostaglandin F_1α level exceeded that of thromboxane B₂ at 8 h postinjury. As examined by inhibitor experiments and northern blotting, cyclooxygenase-1 was responsible for the first phase, and cyclooxygenase-2 was involved in the second phase. On compression injury the levels of interleukin-1α and -1β detected as mRNA and protein increased and peaked at 2-4 h. Injection of exogenous interleukin-1 α into the spinal cord resulted in an increase of cyclooxygenase-2 mRNA content and a predominant production of 6-ketoprostaglandin F_1α resembling the second phase of eicosanoid production. Concomitantly, extravascular migration of polymorphonuclear leukocytes was enhanced after the interleukin-1α injection. These cells together with vascular endothelial cells and glial cells were stained positively with an anti-cyclooxygenase-2 antibody. The results suggest that the immediate eicosanoid synthesis after spinal cord injury was due to the constitutive cyclooxygenase-1 and the delayed synthesis of eicosanoids was attributable to the induction of cyclooxygenase-2 mediated by interleukin-1 α.     

Oxidation of beta-phenylethylamine by both types of monoamine oxidase: effects of substrate concentration and pH.

β-Phenylethylamine (PEA) was characterized as substrate for both type A and type B monoamine oxidase (MAO) in rat brain mitochondria at different substrate concentrations and at different pHs of the reaction media. The experiments on sensitivity to clorygline and deprenyl showed that the inhibition patterns with PEA as substrate differed markedly at different substrate concentrations: at 10 μM, PEA acted as a specific substrate for type B MAO, but at 50–1000 μM it became a common substrate for both types of MAO. The inhibition patterns were also affected markedly by a small change in pH of the reaction medium, especially when PEA concentrations were 50 and 100 μM: the change in pH from 7.2 to 7.8 resulted in the incresse in the proportion of type A MAO by 20–30 per cent. To investigate the mechanisms of such changes in substrate specificity of PEA, kinetic analyses were carried out at pH 7.2 and 7.8 with the uninhibited, the clorgyline-treated (type B) and the deprenyl-treated (type A) enzyme. The Lineweaver-Burk plots for the uninhibited MAO showed strong substrate inhibition for both pHs, which is more marked at pH 7.8 than at pH 7.2. Pretreatment of the enzyme with 10^?7 M clorgyline resulted in generally similar K_m values for PEA to those of the uninhibited enzyme, and the substrate inhibition at pH 7.8 was also stronger than that at pH 7.2. After pretreatment with 10^?7 M deprenyl, the K_m values were higher and the V_max values were lower than those of the uninhibited or the clorgyline-treated enzyme; there was no or only slight substrate inhibition in these curves. These results suggest that the remarkable changes in substrate specificity observed at different PEA concentrations and at different pHs may be due to the strong substrate inhibition of type B MAO.     

Size-dependent mortality of red sea bream, Pagrus major, juveniles released with fluorescent otolith-tags in News Bay, Japan

Totals of 2^.67 x 10⁵ and 7^.56 x 10⁵ juvenile red sea bream of three size groups (10, 20 and 40 mm t.l.) marked with a fluorescent substance in the otolith were released in News Bay, Oita Prefecture, Japan, in July 1987 and June 1988, respectively; the aim was to estimate growth and mortality of different developmental stages.
Of fish released in 1987 and 1988, 10 618 and 4413, respectively were recaptured during those two years. Released fish remained in the bay until the end of summer, and afterwards migrated out towards open waters. Fish of the 40-mm group released in 1987 grew to over 200 mm t.l. in one year. Mean growth rate for 19 days after release was higher in the 40-mm group (0^.87 mm day⁻¹) than in the 20-mm group (0^.74mm day⁻¹). Survival rates over 19 days were 59^.0 and 10^.1 % for 40-mm and 20-mm fish, respectively, in 1987, and those over 30 days were 69^.2, 3^.3 and 0^.0% for 40-mm, 20-mm and 10-mm fish, respectively, in 1988.
Cannibalism was indicated by the presence of marked otoliths for 20-mm fish in the stomachs of a few 40-mm individuals recaptured 2 days after release. Size-dependent growth and size-selective mortality were both noted in juvenile red sea bream, i.e. the relative size differential between larger and smaller individuals was maintained in the period between marking and recapture, and mortality was inversely proportional to size.     

Characterization of [Ca2+]i responses in primary cultures of mouse cardiomyocytes induced by Trypanosoma cruzi trypomastigotes

Trypanosoma cruzi, the protozoan responsible for Chagas disease, employs distinct strategies to invade mammalian host cells. In the present work we investigated the participation of calcium ions on the invasion process using primary cultures of embryonic mice cardiomyocytes which exhibit spontaneous contraction in vitro. Using Fura 2-AM we found that T. cruzi was able to induce a sustained increase in basal intracellular Ca2+ level in heart muscle cells (HMC), the response being associated or not with Ca2+ transient peaks. Assays performed with both Y and CL strains indicated that the changes in intracellular Ca2+ started after parasites contacted with the cardiomyocytes and the evoked response was higher than the Ca2+ signal associated to the spontaneous contractions. The possible role of the extracellular and intracellular Ca2+ levels on T. cruzi invasion process was evaluated using the extracellular Ca2+ chelator EGTA alone or in association with the calcium ionophore A23187. Significant dose dependent inhibition of the invasion levels were found when intracellular calcium release was prevented by the association of EGTA +A23187 in calcium free medium. Dose response experiments indicated that EGTA 2.5 mM to 5 mM decreased the invasion level by 15.2 to 35.1% while A23187 (0.5 M) alone did not induce significant effects (17%); treatment of the cultures with the protease inhibitor leupeptin did not affect the endocytic index, thus arguing against the involvement of leupeptin sensitive proteases in the invasion of HMC.     

Regulatory properties of carbamoyl-phosphate synthetase II from the parasitic protozoan Crithidia fasciculata

1. At the lowered concentrations of 0.5 mM ATP and 1.5 mM MgCl2, 2.0 mM UTP, UDP and UMP inhibited the activity of Crithidia fasciculata carbamoyl-phosphate synthetase II by about 65, 80 and 40% respectively. 2. The result suggests that feedback inhibition of the activity by uridine nucleotides is a mechanism of regulation of the de novo pyrimidine biosynthetic pathway in C. fasciculata. 3. ADP, AMP and CDP inhibited the activity (about 70, 40 and 40%). 4. Excess Mg2+ at around 1 mM, relative to the ATP concentration, was required for the maximum activity. 5. 5-Phosphoribosyl 1-pyrophosphate had no significant effect on the activity under various conditions examined.