MOUSE MUSCLE CELL DIFFERENTIATION IN CLONAL CULTURE

Clonal culture of muscle cells from thigh muscles of the term fetus of the mouse was undertaken. Myoblasts were spherical or spindle-shaped, and proliferated exponentially until day 4 in culture. The generation time of the muscle cells from 2 to 4 days' culture was 9.1 to 13.4 hr. The fusion of myoblasts began on day 4 in culture; many myotubes had been formed by day 6 and spontaneous contraction was observed on day 7. Clonal efficiency was 30%, and the proportion of muscle colonies in all the colonies was 72%.     

INTERACTION OF GIBBERELLIN, KINETIN AND POTASSIUM NITRATE IN THE GERMINATION OF LIGHT-SENSITIVE TOBACCO SEEDS

1. The effects of gibberellin, kinetin, potassium nitrate, andtheir interactions in the germination of light-sensitive tobaccoseeds were studied. 2. Gibberellin was very effective in inducing the dark germinationof tobacco seeds, and a linear relation was found to exist betweenthe germination rate and the concentration of gibberellin ifa suitable temperature was chosen for germination. This linearrelation was, however, changed by illumination and by a raisedtemperature in the manner that the germination on lower concentrationsof gibberellin was increased by the former and decreased bythe latter. 3. Kinetin was effective in promoting the tobacco seed germinationonly when the seeds were irradiated. This light effect was,however, limited only to the longer wavelengths in the visibleregion. The red effect was found to be reversed by infra-redirradiation under the influence of kinetin. 4. The combination of gibberellin and kinetin was synergisticin promoting the tobacco seed germination both in the lightand in the dark, whereas the combination of potassium nitrateand gibberellin or kinetin was synergistic only in the light. (Received November 1, 1960; )     

Dissecting insect responses to climate warming: overwintering and post‐diapause performance in the southern green stink bug,Nezara viridula,under simulated climate‐change conditions

The effect of simulated climate change on overwintering and post‐diapause reproductive performance is studied in Nezara viridula (L.) (Heteroptera: Pentatomidae) close to the species' northern range limit in Japan. Insects are reared from October to June under quasi‐natural (i.e. ambient outdoor) conditions and in a transparent incubator, in which climate warming is simulated by adding 2.5°C to the ambient temperatures. Despite the earlier assumption that females of N. viridula overwinter in diapause, whereas males do so in quiescence, regular dissections show that the two sexes overwinter in a state of true diapause. During winter, both sexes are dark‐coloured and have undeveloped reproductive organs. Resumption of development does not start until late March. During winter, the effect of simulated warming on the dynamics and timing of physiological processes appears to be limited. However, the warming significantly enhances winter survival (from 27–31% to 47–70%), which is a key factor in range expansion of N. viridula. In spring, the effect of simulated warming is complex. It advances the post‐diapause colour change and transition from dormancy to reproduction. The earlier resumption of development is more pronounced in females: in April, significantly more females are already in a reproductive state under the simulated warming than under quasi‐natural conditions. In males, the tendency is similar, although the difference is not significant. Warming significantly enhances spring survival and percentage of copulating adults, although not the percentage of ovipositing females and fecundity. The results suggest that, under the expected climate‐warming conditions, N. viridula will likely benefit mostly as a result of increased winter and spring survival and advanced post‐diapause reproduction. Further warming is likely to allow more adults to survive the critical cold season and contribute (both numerically and by increasing heterogeneity) to the post‐overwintering population growth, thus promoting the establishment of this species in newly‐colonized areas.     

Molecular characterization of microbial communities in fault-bordered aquifers in the Miocene formation of northernmost Japan

We investigated the diversity and distribution of archaeal and bacterial 16S rRNA gene sequences in deep aquifers of mid‐ to late Miocene hard shale located in the northernmost region of the Japanese archipelago. A major fault in the north‐west–south‐east (NW–SE) direction runs across the studied area. We collected three groundwater samples from boreholes on the south‐west (SW) side of the fault at depths of 296, 374 and 625 m below ground level (m.b.g.l.) and one sample from the north‐east (NE) side of the fault at a depth of 458 m.b.g.l. The groundwater samples were observed to be neutral and weakly saline. The total microbial counts after staining with acridine orange were in the order 10⁵?10⁶ cells mL^?1 and 10³ cells mL^?1 in the aquifers to the SW and to the NE of the fault, respectively. A total of 407 archaeal and bacterial 16S rRNA gene sequences (204 and 203 sequences, respectively) were determined for clone libraries constructed from all groundwater samples. Phylogenetic analyses showed that the libraries constructed from the SW aquifers were generally coherent but considerably different from those constructed from the NE aquifer. All of the archaeal clone libraries from the SW aquifers were predominated by a single sequence closely related to the archaeon Methanoculleus chikugoensis, and the corresponding bacterial libraries were mostly predominated by the sequences related to Bacteroidetes, Firmicutes and δ‐Proteobacteria. In contrast, the libraries from the NE aquifer were dominated by uncultured environmental archaeal clones with no methanogen sequences and by β‐proteobacterial clones with no sequences related to Bacteroidetes and δ‐Proteobacteria. Hence, the possible coexistence of methanogens and sulphate reducers in Horonobe deep borehole (HDB) on the SW side is suggested, particularly in HDB‐6 (374 m.b.g.l.). Moreover, these organisms might play an important geochemical role in the groundwater obtained from the aquifers.     

Effect of Extracellular Ions on Motility and Cell Entry in Toxoplasma gondii

Toxoplasma gondii tachyzoites were quiescent in mouse peritoneal fluid or in K₂SO₄ buffer at pH 8.2. They became consistently motile when K⁺ was replaced by other monovalent or divalent cations at a constant pH (pH = 8.2). They also became motile when Cl^? was substituted for SO₄^2-. Nitrate or SCN^?, can also be substituted for Cl^? to a certain extent. Tachyzoites showed independent movement for more than 15 min in KCl, and for about 5 min in the other buffers at pH 8.2 after which they were exhausted and stopped. These tachyzoites could not then be further stimulated to motility by renewal of the suspension buffer. Infection of monolayer cells was demonstrated only with parasites which were motile during inoculation. The highest infectivity was thus obtained either with freshly collected tachyzoites or with those preincubated in K₂SO₄ buffer for 30 min at 37° C at alkaline pH and thus not yet exhausted for motility. Approximately 34 to 38% of these latter organisms were seen to enter cells when they were inoculated into cultures immediately after being resuspended in MEM for 30 min at 37° C. Conversely, those whose motility had been exhausted by the preincubation in buffers other than K₂SO₄, pH 8.2 could not enter monolayer cells. Additionally, parasites were unable to enter cells when inoculated into cultures in K₂SO₄ buffer at alkaline pH; instead they remained quiescent on the surface of the monolayer cells, suggesting that Toxoplasma enters the host cells by active invasion.     

IMMUNOCHEMICAL STUDY ON THE SPECIES SPECIFICITY OF SPERM-BINDING PROTEIN FROM THE SURFACE OF THE SEA URCHIN EGG*

Immunological species specificity of sperm-binding protein from eggs of the 4 sea urchin species, Hemicentrotus pulcherrimus, Pseudocentrotus depressus, Anthocidaris crassispina and Temnopleurus toreumaticus, was examined by means of double immuno-diffusion technique in agar. Ca-soluble fraction of sperm-binding protein which is considered to be responsible for initial sperm-egg bonding at fertilization, has species-specific antigenic component. Correlations in antigenic constituents among the 4 species are described.