The induction of recessive mutations in mouse primordial germ cells with N-ethyl-N-nitrosourea

A specific-locus test was carried out to examine the mutagenic activity of N-ethyl-N-nitrosourea (ENU) on mouse primordial germ cells (PGC). Embryos of C3H/He mice were treated transplacentally with 30 or 50 mg ENU per kg of maternal body weight on day 8.5, 10.5, or 13.5 of gestation (G8.5 day, G10.5 day, or G13.5 day). Male and female mice that had been treated with ENU in embryonic stages were mated with female or male tester PW mice to detect recessive mutations induced in PGC.

ENU induced recessive mutations at a relatively high rate in PGC at these developmental stages. The most sensitive stage was G10.5 day. On G8.5 day, the induced mutation rate in males and females was not significantly different. Cluster mutations, which originate from the limited number of PGC and cell killing, were more frequently induced at an earlier developmental stage. The induced mutation rate per unit dose of ENU (1 mg/kg) was higher in G8.5 and G10.5 day PGC than in stem-cell spermatogonia. It can be concluded that mouse PGC are more sensitive than stem-cell spermatogonia to the induction of recessive mutations by ENU.     

Effects of hydrostatic pressure on the ultrastructure and leakage of internal substances in the yeast Saccharomyces cerevisiae

The structural damage to and leakage of internal substances from Saccharomyces cerevisiae 0–39 cells induced by hydrostatic pressure were investigated. By scanning electron microscopy, yeast cells treated at room temperature with pressuresbellw 400 MPa for 10 min showed a slight alteration in outer shape. Transmission electron microscopy, however, showed that the inner structure of the cell began to be affected, especially the nuclear membrane, when treated with hydrostatic pressure around 100 MPa at room temperature for 10 min; at more than 400–600 MPa, further alterations appeared in the mitochondria and cytoplasm. Furthermore, when high pressure treatment was carried out at — 20° C, the inner structure of the cells was severely damaged even at 200 MPa, and almost all of the nuclear membrane disappeared, although the fluorescent nucleus in the cytoplasm was visible by 4,6-diamidino-2-phenylindole (DAPI) staining. The structural damage of pressure-treated cells was accompanied by the leakage of internal substances. The efflux of UV-absorbing substances including amino acid pools, peptides, and metal ions increased with increase in pressure up to 600 MPa. In particular, amounts of individual metal ion release varied with the magnitude of hydrostatic pressures over 300 MPa, which suggests that the ions can be removed from the yeast cells separately by hydrostatic pressure treatment. Correspondence to: S. Shimada     

A model for neural representation of binocular disparity in striate cortex: distributed representation and veto mechanism

A model in striate cortex is proposed for a distributed neural representation of binocular disparity with a simple cell. In the model, disparity is represented by far, near and tuned inhibitory simple cells. However, the representation will be vetoed by model cells where disparity is excessively large. The veto mechanism consists of a neural network of the model cell which received output from simple cells and which interacts with neighbors. The mechanism is necessary, the model cell responds like a simple cell, and the network is physiologically plausible in the brain. Computer simulation on the neural network model with random dot stereography indicates reasonable performance.     

Inhibition of macrophage nitric oxide production by arachidonate-cascade inhibitors

We examined whether inhibitors of the arachidonic acid cascade inhibited nitric oxide (NO) production, as measured by nitrite concentration, either in macrophages or by their cytosolic fractions. Nitrite production by peritoneal macrophages from mice receiving OK-432 treatment was significantly inhibited by phospholipase A₂ inhibitors [dexamethasone and 4-bromophenacyl bromide (4-BPB)], lipoxygenase inhibitors [nordihydroguaiaretic acid (NDGA) and ketoconazole] and a glutathioneS-transferase (leukotrienes LTA₄-LTC₄) inhibitor (ethacrynic acid). However, caffeic acid and esculetin, inhibitors of 5- and 12-lipoxygenase respectively, were not inhibitory. On the other hand, indomethacin, a cyclooxygenase inhibitor, slightly inhibited whereas another inhibitor, ibuprofen, did not. Inhibition of the nitrite production by dexamethasone, 4-BPB, NDGA and ethacrynic acid was also demonstrated when the macrophages were restimulated ex vivo with OK-432 or with lipopolysaccharide. The inhibitory activity of dexamethasone, NDGA and ethacrynic acid was significantly reduced by ex vivo restimulation with OK-432, whereas that of 4-BPB was hardly affected. Furthermore, the inhibitory activity of dexamethasone, NDGA and ethacrynic acid was much higher when the macrophages were continuously exposed to the agents than when they were pulsed. Meanwhile, inhibition by 4-BPB was almost the same with either treatment. In addition, the inhibitory activity of these agents was not blocked withl-arginine, a substrate of NO synthases, or with arachidonate metabolites (LTB₄, LTC₄ and LTE₄). Ethacrynic acid and 4-BPB, but not dexamethasone and NDGA, also inhibited nitrite production by the cytosolic fractions from OK-432-restimulated peritoneal macrophages, and the inhibitory activity of 4-BPB was superior to that of ethacrynic acid. These agents, however, did not inhibit nitrite production from sodium nitroprusside, a spontaneous NO-releasing compound. These results indicate that dexamethasone, 4-BPB, NDGA and ethacrynic acid inhibited the production of NO by macrophages through at least two different mechanisms: one was inhibited by dexamethasone, NDGA and ethacrynic acid and the other by 4-BPB. Furthermore, 4-BPB and ethacrynic acid directly inhibited the activity of the NO synthase in macrophages, suggesting that the agents work by binding to the active site(s) of the enzyme.     

A protein fraction from aged garlic extract enhances cytotoxicity and proliferation of human lymphocytes mediated by interleukin-2 and concanavalin A

Fraction 4 (F4), a protein fraction isolated from aged garlic extract, enhanced cytotoxicity of human peripheral blood lymphocytes (PBL) against both naturalkiller (NK)-sensitive K562 and NK-resistant M14 cell lines. Although F4 treatment alone increased cytotoxicity, the effect was more remarkable when F4 was administered together with suboptimal doses of interleukin-2 (IL-2); combination treatment of 5 g/ml F4 plus 10 U/ml IL-2 for 72 h generated lymphokine-activated killer activity equivalent to that produced by 100 U/ml IL-2 alone against M14. F4 enhanced IL-2-induced proliferation and IL-2 receptor (Tac) expression of PBL without significant increase of IL-2 production. The enhancement of cytotoxicity both by F4 alone and by F4 plus IL-2 was abolished by anti-IL-2 antibody. F4 also enhanced concanavalin-A(ConA)-induced proliferation of PBL. Radiolabeled-ConA binding assays revealed that F4 treatment greatly augmented the affinity and slightly increased the number of ConA binding sites in PBL. F4 also enhanced ConA-induced IL-2 receptor (Tac) expression and IL-2 production of PBL. Anti-IL-2 antibody inhibited the effect of F4 on ConA-induced proliferation. These data suggest that IL-2 is involved in augmentative effects of F4. Our results indicate that F4 is a very efficient immunopotentiator and may be used for immunotherapy.     

Enantioselective pharmacokinetics of homochlorcyclizine: III. Simultaneous determination of (+)- and (−)- homochlorcyclizine in human urine by high-performance liquid chromatography

A method is described for the simultaneous determination of (+)- and (−)-homochlorcyclizine (HCZ) in human urine by high-performance liquid chromatography on a chiral stationary phase of ovomucoid-bonded silica. The pH of the buffer and organic modifier in the mobile phase markedly affected the chromatographic separation. A mobile phase of methanol—0.02 M acetate buffer (pH 4.7) (25:75, v/v) at a flow-rate of 1.0 ml/min was used for the urine assays. The ultraviolet absorption was monitored at 240 nm, and diphenhydramine was employed as the internal standard for the quantitation. (+)-HCZ, (−)-HCZ and the internal standard were eluted at retention times of 15, 25 and 8 min, respectively. The limit of determination for HCZ enantiomers was ca. 50 ng/ml of urine. One of the metabolites in human urine, which was a quaternary ammonium-linked glucuronide, could also be determined in a manner similar to unchanged HCZ after β-glucuronidase hydrolysis. A pharmacokinetic study was conducted with three healthy volunteers, who each received a single oral dose of racemic HCZ (20 mg). Distinct differences were found between the two enantiomers, particularly in the metabolic process, that is, the urinary excretion as (−)-HCZ-glucuronide within 48 h was ca. four times higher than that of the (+)-isomer. This method should be very useful for enantioselective pharmacokinetic studies of HCZ.