Kenichi Shibata

Collective and individual glycolytic oscillations in yeast cells encapsulated in alginate microparticles

Yeast cells were encapsulated into alginate microparticles of a few hundred micrometers diameter using a centrifuge-based droplet shooting device. We demonstrate the first experimental results of glycolytic oscillations in individual yeast cells immobilized in this way. We investigated both the individual and collective oscillatory behaviors at different cell densities. As the cell density increased, the amplitude of the individual oscillations increased while their period decreased, and the collective oscillations became more synchronized, with an order parameter close to 1 (indicating high synchrony). We also synthesized biphasic-Janus microparticles encapsulating yeast cells of different densities in each hemisphere. The cellular oscillations between the two hemispheres were entrained at both the individual and population levels. Such systems of cells encapsulated into microparticles are useful for investigating how cell-to-cell communication depends on the density and spatial distribution of cells.

Primordial oscillations in life: Direct observation of glycolytic oscillations in individual HeLa cervical cancer cells

We report the first direct observation of glycolytic oscillations in HeLa cervical cancer cells, which we regard as primordial oscillations preserved in living cells. HeLa cells starved of glucose or both glucose and serum exhibited glycolytic oscillations in nicotinamide adenine dinucleotide (NADH), exhibiting asynchronous intercellular behaviors. Also found were spatially homogeneous and inhomogeneous intracellular NADH oscillations in the individual cells. Our results demonstrate that starved HeLa cells may be induced to exhibit glycolytic oscillations by either high-uptake of glucose or the enhancement of a glycolytic pathway (Crabtree effect or the Warburg effect), or both. Their asynchronous collective behaviors in the oscillations were probably due to a weak intercellular coupling. Elucidation of the relationship between the mechanism of glycolytic dynamics in cancer cells and their pathophysiological characteristics remains a challenge in future.

The effects of starvation and acidification on lag phase duration of surviving yeast cells

Starvation is one of the most common forms of stress experienced in the wild life. Such conditions associate the other forms of stress such as acid, heat, oxidation, and so on. Organisms acclimate to such stresses and acquire the stress tolerances, which often trade-off their growth rates. To investigate whether starvation and the associated stresses may cause the changes in the growth and the central carbon metabolism, we stock-cultured the yeast S. cerevisiae on YNB agar plates up to a month and subsequently cultured in YNB broth. The pH of the agar medium just under the yeasts colonies sharply dropped from 5.0 to 3.9 in the first day, eventually reached approximately 3.0, and the viability logarithmically decreased. The surviving cells accumulated cell damages that were measured as the prolonged LPDs (lag phase durations). We did not, however, observe the effects of long-term stock-cultivations on the measured phenotypes: growth rates, the carrying capacities, and the glycolytic oscillations that are the temporal dynamics of the central carbon metabolism. Our study revealed that the contribution of cell damages to the total delay in growth was 78%, and that LPDs are closely related to damage-recovery mechanisms.

Lower sensitivity of cyprinid fishes to three acetylcholinesterase inhibitor pesticides: an evaluation based on no-effect concentrations

Researchers have suggested that cyprinid fishes are less sensitive to chemical stress than comparable fish families, yet few empirically based evaluations of this hypothesis have been conducted. In this study, we developed a generalized linear mixed model in which the no-effect concentrations (NECs; threshold concentration below which no effect on survival is predicted during prolonged exposure) of 29 fish species from 13 families exposed to an acetylcholinesterase inhibitor pesticide (carbaryl, chlorpyrifos, or malathion) were used as the response variable. The corresponding specific somatic maintenance (SSM) rates, as a size-independent proxy for fish metabolism and a categorical variable regarding whether the species is a cyprinid, were used as the predictor variables. We included SSM rates in the analysis because previous work demonstrated that they are negatively correlated with NECs. Our results indicate that the NECs for cyprinid fishes were significantly higher than those for other fishes, suggesting that cyprinids are indeed less sensitive to the three studied pesticides. Although the SSM rates were negatively related with the NECs, the actual relationship between the two was not clear, implying that the importance of SSM rates may depend on the taxonomic group tested.

Promotion and inhibition of synchronous glycolytic oscillations in yeast by chitosan

Synchronous rhythmic activities play crucial roles in diverse biological systems. Glycolytic oscillations in yeast cells have been studied for 50 years with the aim of elucidating the mechanisms underlying the intracellular oscillations and their synchronization. We investigated the effects of chemical disturbances on the individual and collective glycolytic oscillations in yeast cells encapsulated in alginate microparticles, and demonstrated that the addition of chitosan, an antimicrobial agent, decreased the duration of these oscillations. In contrast, the periods and the synchronicity states showed two different responses to the chitosan treatments. The periods were shown to be prolonged following the treatment with 5–50 mg·L−1 and shortened at 75 mg·L−1 of chitosan. Collective oscillations became more synchronized at 5 mg·L−1 of chitosan, and desynchronized at 25–75 mg·L−1 of this compound. These findings can be explained by the balance between two chitosan features, increasing cell membrane permeability and acetaldehyde scavenging. At low concentrations, chitosan presumably acts as a synchronization promoter that does not mediate the synchronization itself but induces an increase in intercellular coupling. We believe that our findings may provide new insights into the synchronous rhythmic activities in biological systems.