Spin Trapping Isotopically-Labelled Nitric Oxide Produced from [15n]L-Arginine and [17ojdioxygen by Activated Macrophages Using a Water Soluble Fe++-Dithiocarbamate Spin Trap

The unique capabilities of EPR spin trapping of nitric oxide based on a ferrous-dithiocarbamate spin trap have been demonstrated in a study verifying the source of the nitrogen and oxygen atoms in nitric oxide produced from activated macrophages. Spin trapping experiments were performed during nitric oxide generation from activated mouse peritoneal macrophages using the ferrous complex of N-methyl D-glucam-ine dithiocarbamate as a spin trap. When ¹⁵N-substituted arginine was given to the activated macrophages in the presence of the spin trap, a characteristic EPR spectrum of the nitric oxide spin adduct was obtained, which indicates the presence of the ^l5N atom in the nitric oxide molecule. The hyperfine splitting (hfs) constant of the ^l5N nucleus was 17.6 gauss. When ^l7O-containing dioxygen (55%) was supplied to the medium, an EPR spectrum consistent with the “O-substituted nitric oxide spin adduct was observed in the composite spectrum. The hfs of “O was estimated to be 2.5 gauss. The ^l4NO spin adduct observed after prolonged incubation in the medium which contains [^l5N]L-arginine as the only extracellular source of arginine demonstrates that arginine is recycled through its metabolite in activated macrophages.     

Nanoparticles Containing Curcumin Useful for Suppressing Macrophages In Vivo in Mice

To explore a novel method using liposomes to suppress macrophages, we screened food constituents through cell culture assays. Curcumin was one of the strongest compounds exhibiting suppressive effects on macrophages. We subsequently tried various methods to prepare liposomal curcumin, and eventually succeeded in preparing liposomes with sufficient amounts of curcumin to suppress macrophages by incorporating a complex of curcumin and bovine serum albumin. The diameter of the resultant nanoparticles, the liposomes containing curcumin, ranged from 60 to 100 nm. Flow cytometric analyses revealed that after intraperitoneal administration of the liposomes containing curcumin into mice, these were incorporated mainly by macrophages positive for F4/80, CD36, and CD11b antigens. Peritoneal cells prepared from mice injected in vivo with the liposomes containing curcumin apparently decreased interleukin-6-producing activities. Major changes in body weight and survival rates in the mice were not observed after administrating the liposomes containing curcumin. These results indicate that the liposomes containing curcumin are safe and useful for the selective suppression of macrophages in vivo in mice.     

Aminoglutethimide effect on circadian rhythms in urinary variables in a patient with idiopathic hyperaldosteronism

Aminoglutethimide (AG: 750 mg/day) was administered to a patient with idiopathic hyperaldosteronism (IHA) and circadian rhythms in urinary excretion of sodium (UNaV), potassium (UKV), aldosterone (AER) and 17-OHCS were analyzed by the single cosinor method. Urine was collected every 4h for 24h on the day before and on the 1st, 3rd and 7th day of AG administration, and above variables in each sample were determined. Circadian rhythms of 14 patients with primary aldosteronism (PA) who served as controls were also analyzed. In the present case, circadian acrophases in UNaV and AER studied before AG administration occurred at 22(19) and 07(05), respectively. They were similar to those of preoperative PA-patients. Circadian acrophase in UNaV occurred earlier with AG administration and on the 7th day it was at 14(05), a value similar to that of postoperative PA-patients. Circadian mesor in AER decreased remarkably from 4.1 to 0.6 micrograms/4h with AG administration, as did circadian mesor in UKV, whereas circadian mesor and acrophase in 17-OHCS did not change. Thus, the circadian characteristics in urinary variables in the present IHA-case were pathophysiologically similar to those of PA.     

Host concealment: a determinant for host acceptance and feeding in an ectoparasitoid wasp

In general, ectoparasitoids attack concealed hosts in protected situations whereas endoparasitoids use both concealed and exposed hosts. The difference is assumed to be the consequence of ecological constraints; ectoparasitic larvae are vulnerable both to predation and to climatic factors such as rainfall, and, hence, require some structures to protect themselves. I hypothesized that such ecological constraints should act as a within-species selection pressure to female ectoparasitoids, and hence that females should recognize the degree of concealment of the host and prefer concealed over exposed hosts for oviposition. To test this hypothesis, I examined 1) whether host concealment could influence host acceptance by the ectoparasitoid wasp Agrothereutes lanceolatus and 2) whether host concealment could influence the fitness of the offspring. Female wasps recognized and attacked (probed) both cocooned and exposed host prepupae in equal proportions, but discriminated between them after ovipositor insertion, and preferred the former for oviposition and the latter for host-feeding. They also selected to oviposit on hosts concealed in paper tubes. Thus host concealment was important for host selection in A. lanceolatus . Offspring fitness (measured as survival and size) was much lower on exposed hosts than on cocooned and paper-concealed hosts, even under laboratory conditions. Thus, host concealment influenced the fitness of wasp offspring, and, hence, is a good indicator of host quality for female wasps. Adaptiveness of host selection and host-feeding in A. lanceolatus in relation to host concealment is discussed.     

The effect of sperm preparation and co-incubation time on in vitro fertilization of Bos indicus oocytes.

The objective of the present study was to evaluate the effect of various methods of sperm selection and various sperm-oocyte co-incubation times on in vitro fertilization (IVF) of zebu (Bos indicus) oocytes. Frozen semen from one ejaculate of a single bull was used for all treatments and replicates. After thawed, sperm was subjected to one of the three treatments: 45 and 90% discontinuous Percoll gradient, swim-up and washing by centrifugation. In all treatments, the spermatozoa were incubated with in vitro matured oocytes for 3, 6, 12 and 18h. After co-incubation oocytes were transferred to the culture medium and culture for 44h, when the cleavage was evaluated. The uncleavaged oocytes were fixed and stained to determine penetration, pronucleus formation and polyspermy. The sperm selection method did not influence (P<0.05) polyspermy, pronucleus formation, penetration and cleavage rates. No interaction between method of selection and sperm-oocyte co-incubation time was observed (P>0.05). However, sperm-oocyte co-incubation time affected fertilization. The lower penetration (26.5%) and cleavage rates (13.1%) were obtained at 3-h period. The penetration and cleavage percentages increased (P<0.05) progressively at 6h (63.3 and 54.4%) and 12h (77.6 and 67.6%). No differences (P>0.05) were observed between 12 and 18h of incubation for penetration and cleavage rates. The incidence of polyspermy and pronucleus formation was similar (P>0.05) for all time points. It is concluded that the methods used in this study for sperm selection do not affect fertilization; therefore, they all can be used for bovine IVF. In addition, regardless the method used better fertilization results were obtained when sperm and oocytes were co-incubated for 12h, and the prolongation of that time for up to 18h had no detrimental effect on fertilization.     

Involvement of aquaporin in thromboxane A2 receptor-mediated, G12/13/RhoA/NHE-sensitive cell swelling in 1321N1 human astrocytoma cells

The physiological role of the thromboxane A₂ (TXA₂) receptor expressed on glial cells remains unclear. We previously reported that 1321N1 human astrocytoma cells pretreated with dibutyryl cyclic AMP (dbcAMP) became swollen in response to U46619, a TXA₂ analogue. In the present study, we examined the detailed mechanisms of TXA₂ receptor-mediated cell swelling in 1321N1 cells. The cell swelling caused by U46619 was suppressed by expression of p115-RGS, an inhibitory peptide of Gα_12/13 pathway and C3 toxin, an inhibitory protein for RhoA. The swelling was also inhibited by treatment with Y27632, a Rho kinase inhibitor and 5-(ethyl-N-isopropyl)amiloride (EIPA), a Na⁺/H⁺-exchanger inhibitor. Furthermore, cell swelling was suppressed by the pretreatment with aquaporin inhibitors mercury chloride or phloretin in a concentration-dependent manner, suggesting that aquaporins are involved in U46619-induced 1321N1 cell swelling. In fact, U46619 caused [³H]H₂O influx into the cells, which was inhibited by p115-RGS, C3 toxin, EIPA, mercury chloride and phloretin. This is the first report that the TXA₂ receptor mediates water influx through aquaporins in astrocytoma cells via TXA₂ receptor-mediated activation of Gα_12/13, Rho A, Rho kinase and Na⁺/H⁺-exchanger.     

Structure and function of muscle myosin

We have studied the primary structures of myosins from chicken muscles in order to clarify the relationship between structure and function of muscle myosin. The primary structures of the various kinds of light chains from chicken muscle myosins have been determined. We also report the primary structure of the 23K fragment of subfragment-1 (S-1) component from the heavy chain of chicken fast skeletal muscle myosin. In addition, antibody was prepared against the 23K fragment. The antibody was found to inhibit the Mg²⁺-ATPase activity and the initial P_i burst of the ATPase in the S-1 component. The antibody suppressed the ATP-induced fluorescence enhancement of S-1, though it did not suppress the binding of ATP to S-1. These results are also discussed.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.