The polarity of fucoid eggs is fixed either when tip growth starts or a bit earlier. A steady flow of calcium ions into the incipient tip is thought to establish a high calcium zone that is needed for its localization and formation. To test this hypothesis, we have injected seven different 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-type calcium buffers into Pelvetia eggs many hours before tip growth normally starts. Critical final cell concentrations of each buffer prove to block outgrowth (as well as cell division) for up to 2 weeks. This critical inhibitory concentration is lowest for two buffers with dissociation constants or Kd values of 4-5 x 10(-6) M and increases steadily as the buffers' Kd values shift either below or above this optimal value to ones as low as 4 x 10(-7) M or as high as 9.4 x 10(-5) M. To analyze these results, we have derived an equation (based on the concept of facilitated diffusion) for the effects of diffusable calcium buffers on steady-state calcium gradients. The data fit this equation quite well if it is assumed that cytosolic free calcium at the incipient tip is normally kept at about 7 microM and, thus, far above the general cytosolic level.

The prokaryotic Synechococcus sp. RF-1 exhibited a nitrogen fixation circadian rhythm with characteristics remarkably similar to the circadian rhythm of eukaryotes. The rhythm had a freerunning period of about 24 hours when the length of the preentrained cycle did not differ too much from 24 hours, and it was insensitive to changes in temperature from 220C to 330C. Because the endogenous rhythm of nitrogen fixation was not affected by a phase-shift of its previous cycles, the circadian rhythm in Synechococcus sp. RF-1 was not considered to be controlled simply by a feedback mechanism.medium (13)

In the prokaryote Synechococcus RF-1, circadian changes in the uptake of L-leucine and 2-amino isobutyric acid were observed. Uptake rates in the light period were higher than in the dark period for cultures entrained by 12/12 hour light/dark cycles. The periodic changes in L-leucine uptake persisted for at least 72 hours into continuous light (L/L). The rhythm had a freerunning period of about 24 hours in L/L at 290C. A single dark treatment of 12 hours could initiate rhythmic leucine uptake in an L/L culture. The phase of rhythm could be shifted by a pulse of low temperature (0°C). The free-running periodicity was "temperature-compensated" from 21 to 370C. A 24 hour depletion of extracellular Ca2+ before the free-running L/L condition reduced the variation in uptake rate but had little effect on the periodicity of the rhythm. The periodicity was also not affected by the introduction of 25 mm NaNO3. The uptake rates for 20 natural amino acids were studied at 12 hour intervals in cultures exposed to 12/12 hour light/dark cycles. For eight of these amino acids Circadian rhythms have been documented for organisms throughout the eukaryotic kingdoms (3). Circadian rhythmicity had not been detected in any prokaryote until recently, when endogenous circadian timing in nitrogen-fixation (5,8,12) and cell division (12,16) were reported in some strains of unicellular cyanobacteria belonging to the genus Svnechococcus.In eukaryotic organisms, endogenous circadian changes in the uptake rates of histidine and lysine have been observed in the yeast Saccharomyces cerevisiae (4).In this report, we investigated the uptake rates of natural amino acids in the prokaryotic Synechococcus RF-1 in an attempt to observe endogenous circadian variations. For comparison, we also investigated uptake rates of L-leucine in Svnechococcuis PCC 7942 and Synechocystis PCC 6803. MATERIALS AND METHODSSvnechococcus RF-1 (PCC 8801) was cultured as described previously (8 When cells of Synechococcus RF-1 were cultured under 12/ 12 h L/D cycles, the uptake rates of leucine (Fig. 1A) and non-metabolizable AIB (Fig. 2) fluctuated periodically and were several times higher during the light period than during the dark period. After the cultures were subsequently exposed to L/L, the periodic variations in leucine and AIB uptake persisted for at least 72 h (Fig. 1B) and 60 h ( Fig. 2A), respectively, without a noticeable change in periodicity. The average rhythm period under free-running conditions was about 24 h. The amino acid uptake rhythm was about 12 h out of phase with the nitrogen-fixation (5,8)

Japanese encephalitis virus (JEV) is among the major threats to public health in Asia. For disease control and prevention, the efficient production of safe and effective vaccines against JEV is in urgent need. In this study, we produced a plant-made JEV vaccine candidate using a chimeric virus particle (CVP) strategy based on bamboo mosaic virus (BaMV) for epitope presentation. The chimeric virus, designated BJ2A, was constructed by fusing JEV envelope protein domain III (EDIII) at the N-terminus of BaMV coat protein, with an insertion of the foot-and-mouth disease virus 2A peptide to facilitate the production of both unfused and epitope-presenting for efficient assembly of the CVP vaccine candidate. The strategy allowed stable maintenance of the fusion construct over long-term serial passages in plants. Immuno-electron microscopy examination and immunization assays revealed that BJ2A is able to present the EDIII epitope on the surface of the CVPs, which stimulated effective neutralizing antibodies against JEV infection in mice. This study demonstrates the efficient production of an effective CVP vaccine candidate against JEV in plants by the BaMV-based epitope presentation system.