Transformation of L cells with virus thymidine kinase genes introduced by red cell-mediated microinjection

Fusion of red cell ghosts containing foreign materials with cells results in the introduction of the materials into the cells (red cell-mediated microinjection). Until now, 'two-step dialysis' has mainly been used for trapping proteins in the ghosts. Large-sized materials such as DNA, however, are rarely trapped in the ghosts, since the holes in the red cell membrane caused by osmotic shock are too small for such materials to pass through. In this study, we improved the trapping technique. Some of the Hind III fragments of lambda phage DNA as well as proteins could be trapped in the ghosts when the mixture of these materials and red cells were frozen at -80 degrees C for a short period followed by quick thawing. Red cell-mediated microinjection using ghosts containing plasmid pBR322 linked with a Herpes simplex viral thymidine kinase (tk) gene brought about transformation of tk-defective L cells, the efficiency of transformation was 1 out of 20 000-60 000 cells fused with the ghosts.     

Spatio-temporal distribution patterns of two tachinid flies,Epicampocera succincta andCompsilura concinnata parasitizingPieris

Larvae of genus Pieris in the northern part of Kyoto City are parasitized by two tachinid flies:Epicampocera succincta, a specialist on genus Pieris, and Compsilura concinnata, a generalist with very wide host-range. We surveyed the parasitism rates of Pieris by both flies for two years at six study areas. In these study areas, there lived three host species in the genus Pieris: P. rapae, P. melete, and P. napi, but neither tachinid parasitized P. napi to any significant extent. In the mountainous district, P. rapae and P. melete coexisted and their populations were relatively continuous, while in the lowland, only P. rapae larvae were abundant in spring and autumn, but even they disappeared in summer. Parasitisms by E. succincta occurred mainly in mountainous district and never in the lowland. C. concinnata parasitized Pieris in all the areas, but its parasitisms occurred mainly in autumn. We analyzed the factors affecting the spatial and temporal patterns of parasitism rates and presumed that the temporal discontinuity of host population restricted the distribution of the specialist parasitoid.     

Activation of Xenopus Eggs by an Extract of Cynops Sperm

An extract obtained from Cynops sperm induced the activation of both Cynops and Xenopus eggs with accompanying changes in the potential of the egg membrane that were quite similar to those caused by the Cynops sperm. The activation-inducing properties of the extract were abolished by treatment with proteinase K or by heating (60°C, 15 min) and were associated with a protease activity against peptidyl Arg-MCA substrates. The activation of Xenopus eggs by the extract was inhibited by those substrates, or by protease inhibitors, aprotinin or leupeptin. The protease activity was localized in the acrosomal region of Cynops sperm. The activation of Xenopus eggs by the extract was prevented when the exterior concentration of Ca²⁺ions, [Ca²⁺]₀, was reduced to 1.5 μM, but it was enhanced when [Ca²⁺]₀ was increased to 340 μM. The activation of Xenopus eggs by the extract was not affected by positive clamping when [Ca²⁺]₀ was 340 μM. These results suggest that the sperm extract contains a protease that causes an increase in the influx of Ca²⁺ions that results in voltage-insensitive activation of the egg.     

Guest-induced conformational change of β-cyclodextrin capped with an environmentally sensitive chromophore

A capped cyclodextrin, 6-deoxy-6-(p-hydroxy-m-nitrophenacylthio)-β-cyclodextrin, was prepared in order to detect any conformational change of the host upon the guest binding. The association constant between the cyclodextrin and 1-adamantanecarboxylate, cyclohexanecarboxylate, p-methylbenzoate, 3,3-dimethylbutyrate, or 2,2-dimethylpropionate was enhanced 20, 5.3, 3.7, 2.3, or 2.0 times, respectively, by chromophore capping. The changes in the electronic, NMR, and circular dichroism spectra as well as pK_a of this cyclodextrin upon binding of the guest strongly indicate a conformational change around the chromophoric moiety of the cyclodextrin.     

Characterization of glutamate transport system in hydrophobic protein (H protein) of Bacillus subtilis

Hydrophobic protein (H protein) was isolated from membrane fractions of Bacillus subtilis and constituted into artificial membrane vesicles with lipid of B. substilis. Glutamate was accumulated into the vesicle when a Na⁺ gradient across the membrane was imposed. The maximum effect of Na⁺ on the transport was achieved at a concentration of about 40 mM, while the apparent K_m for Na⁺ was approximately 8 mM. On the other hand, K_m for glutamate in the presence of 50 mM Na⁺ was about 8 μM. Increasing the concentration of Na⁺ resulted in a decrease in K_m for glutamate, maximum velocity was not affected. The transport was sensitive to monensin (Na⁺ ionophore).Glutamate was also accumulated when pH gradient (interior alkaline) across the membrane was imposed or a membrane potential was induced with K⁺-diffusion potential. The pH gradient-driven glutamate transport was sensitive to carbonylcyanide m-chlorophenylhydrazone and the apparent K_m for glutamate was approximately 25 μM.These results indicate that two kinds of glutamate transport system were present in H protein: one is Na⁺ dependent and the other is H⁺ dependent.     

On a method for estimating the rate of population interchange between two areas

A method for estimating the rate of population interchange between two areas is described in this paper. It is applicable to the case where the sampling ratios and the survival factors of the populations on two areas are different from each other. From the marking-and-recapture data taken at three successive occasions, we can obtain the estimates of emigration factors, total survival factors over two areas and the ratio of surviving migrants to the total survivors over the two areas, for each of two populations initially living on different areas.