Novel biochemical manipulation of brain serotonin reveals a role of serotonin in the circadian rhythm of sleep-wake cycles.

Serotonin (5-HT) neurons have been implicated in the modulation of many physiological functions, including mood regulation, feeding, and sleep. Impaired or altered 5-HT neurotransmission appears to be involved in depression and anxiety symptoms, as well as in sleep disorders. To investigate brain 5-HT functions in sleep, we induced 5-HT deficiency through acute tryptophan depletion in rats by intraperitoneally injecting a tryptophan-degrading enzyme called tryptophan side chain oxidase I (TSOI). After the administration of TSOI (20 units), plasma tryptophan levels selectively decreased to 1-2% of those of controls within 2 h, remained under 1% for 12-24 h, and then recovered between 72 and 96 h. Following plasma tryptophan levels, brain 5-HT levels decreased to ∼30% of the control level after 6 h, remained at this low level for 20-30 h, and returned to normal after 72 h. In contrast, brain norepinephreine and dopamine levels remained unchanged. After TSOI injection, the circadian rhythms of the sleep-wake cycle and locomotive activity were lost and broken into minute(s) ultradian alternations. The hourly slow-wave sleep (SWS) time significantly increased at night, but decreased during the day, whereas rapid eye movement sleep was significantly reduced during the day. However, daily total (cumulative) SWS time was retained at the normal level. As brain 5-HT levels gradually recovered 48 h after TSOI injection, the circadian rhythms of sleep-wake cycles and locomotive activity returned to normal. Our results suggest that 5-HT with a rapid turnover rate plays an important role in the circadian rhythm of sleep-wake cycles.

Quantitative analysis of the bacteriophage Qbeta infection cycle.

In this study, the infection cycle of bacteriophage Qbeta was investigated. Adsorption of bacteriophage Qbeta to Escherichia coli is explained in terms of a collision reaction, the rate constant of which was estimated to be 4x10(-10) ml/cells/min. In infected cells, approximately 130 molecules of beta-subunit and 2x10(5) molecules of coat protein were translated in 15 min. Replication of Qbeta RNA proceeded in 2 steps-an exponential phase until 20 min and a non-exponential phase after 30 min. Prior to the burst of infected cells, phage RNAs and coat proteins accumulated in the cells at an average of up to 2300 molecules and 5x10(5) molecules, respectively. An average of 90 infectious phage particles per infected cell was released during a single infection cycle up to 105 min.

A novel sequence-specific RNA quantification method using nicking endonuclease, dual-labeled fluorescent DNA probe, and conformation-interchangeable oligo-DNA.

We have developed a novel, single-step, isothermal, signal-amplified, and sequence-specific RNA quantification method (L-assay). The L-assay consists of nicking endonuclease, a dual-labeled fluorescent DNA probe (DL-probe), and conformation-interchangeable oligo-DNA (L-DNA). This signal-amplified assay can quantify target RNA concentration in a sequence-specific manner with a coefficient of variation (Cv) of 5% and a lower limit of detection of 0.1 nM. Moreover, this assay allows quantification of target RNA even in the presence of a several thousandfold excess by weight of cellular RNA. In addition, this assay can be used to measure the changes in RNA concentration in real-time and to quantify short RNAs (<30 nucleotides). The L-assay requires only incubation under isothermal conditions, is inexpensive, and is expected to be useful for basic research requiring high-accuracy, easy-to-use RNA quantification, and real-time quantification.

Importance of translation-replication balance for efficient replication by the self-encoded replicase.

In all living systems, the genetic information is replicated by the self-encoded replicase (Rep); this can be said to be a self-encoding system. Recently, we constructed a self-encoding system in liposomes as an artificial cell model, consisting of a reconstituted translation system and an RNA encoding the catalytic subunit of Qbeta Rep and the RNA was replicated by the self-encoded Rep produced by the translation reaction. In this system, both the ribosome (Rib) and Rep bind to the same RNA for translation and replication, respectively. Thus, there could be a dilemma: effective RNA replication requires high levels of Rep translation, but excessive translation in turn inhibits replication. Herein, we actually observed the competition between the Rib and Rep, and evaluated the effect for RNA replication by constructing a kinetic model that quantitatively explained the behavior of the self-encoding system. Both the experimental and theoretical results consistently indicated that the balance between translation and replication is critical for an efficient self-encoded system, and we determined the optimum balance.

Replication of genetic information with self-encoded replicase in liposomes.

In all living systems, the genome is replicated by proteins that are encoded within the genome itself. This universal reaction is essential to allow the system to evolve. Here, we have constructed a simplified system involving encapsulated macromolecules termed a "self-encoding system", in which the genetic information is replicated by self-encoded replicase in liposomes. That is, the universal reaction was reconstituted within a microcompartment bound by a lipid bilayer. The system was assembled by using one template RNA sequence as the information molecule and an in vitro translation system reconstituted from purified translation factors as the machinery for decoding the information. In this system, the catalytic subunit of Qbeta replicase is synthesized from the template RNA that encodes the protein. The replicase then replicates the template RNA that was used for its production. This in-liposome self-encoding system is one of the simplest such systems available; it consists of only 144 gene products, while the information and the function for its replication are encoded on different molecules and are compartmentalized into the microenvironment for evolvability.

Symbiotic bacteria contribute to increasing the population size of a freshwater crustacean, Daphnia magna.

The filter-feeding crustacean Daphnia is a key organism in freshwater ecosystems. Here, we report the effect of symbiotic bacteria on ecologically important life history traits, such as population dynamics and longevity, in Daphnia magna. By disinfection of the daphniid embryos with glutaraldehyde, aposymbiotic daphniids were prepared and cultured under bacteria-free conditions. Removal of bacteria from the daphniids was monitored by quantitative polymerase chain reaction for bacterial 16S rRNA gene. The population of aposymbiotic daphniids was reduced 10-folds compared with that of the control daphniids. Importantly, re-infection with symbiotic bacteria caused daphniids to regain bacteria and increase their fecundity to the level of the control daphniids, suggesting that symbiotic bacteria regulate Daphnia fecundity. To identify the species of symbiotic bacteria, 16S rRNA genes of bacteria in daphniids were sequenced. This revealed that 50% of sequences belonged to the Limnohabitans sp. of the Betaproteobacteria class and that the diversity of bacterial taxa was relatively low. These results suggested that symbiotic bacteria have a beneficial effect on D. magna, and that aposymbiotic Daphnia are useful tools in understanding the role of symbiotic bacteria in the environmental responses and evolution of their hosts.

Enzymatic depletion of serotonin in vivo and its consequences.

The serotonin (5-HT) system in the brain is a global modulator thought to tune up a unique subset of brain keynotes such as emotion, motivation, and sleep/conscious states in the deepest seats of cognition and behavior. In pursuit for coherent accounts of such higher-order issues, we have been trying to deduce the system dynamics of 5-HT in full span from the perturbation-response couples elicited by our most quick, specific, extensive, and reversible depletion of the brain 5-HT so far available via plasma precursor annihilation by injection of a tryptophan degrading enzyme (TSO) (ISTRY meetings-1986 in Cardiff, -92 in Nagoya and -98 in Hamburg). Herein discussed are the dynamics of the 5-HT depletion both in the whole brain and regional dimensions, and then the perturbation-induced manifestation of a continuous behavioral quiescence underlain by chaotic patterns of sleep/waking states. This response in sharp contrast to those by earlier serotonin depletors, prompts us to consider a serious revision of the current 5-HT scenario. In the light of our research, future directions will be discussed together with the RTD (rapid tryptophan depletion) claiming the impaired brain 5-HT turnover by a partial decline of plasma tryptophan.

Kinetic analysis of the entire RNA amplification process by Qbeta replicase.

The kinetics of the RNA replication reaction by Qbeta replicase were investigated. Qbeta replicase is an RNA-dependent RNA polymerase responsible for replicating the RNA genome of coliphage Qbeta and plays a key role in the life cycle of the Qbeta phage. Although the RNA replication reaction using this enzyme has long been studied, a kinetic model that can describe the entire RNA amplification process has yet to be determined. In this study, we propose a kinetic model that is able to account for the entire RNA amplification process. The key to our proposed kinetic model is the consideration of nonproductive binding (i.e. binding of an enzyme to the RNA where the enzyme cannot initiate the reaction). By considering nonproductive binding and the notable enzyme inactivation we observed, the previous observations that remained unresolved could also be explained. Moreover, based on the kinetic model and the experimental results, we determined rate and equilibrium constants using template RNAs of various lengths. The proposed model and the obtained constants provide important information both for understanding the basis of Qbeta phage amplification and the applications using Qbeta replicase.

Femtoliter compartment in liposomes for in vitro selection of proteins.

The aqueous compartment in liposomes provides a reaction resembling the cell and therefore is used as a microcompartment in which to study enzymatic reactions. However, regardless of their method of preparation, the heterogeneity in size of cell-size liposomes limits their potential uses. We established a strategy to estimate the internal aqueous volume of cell-size liposomes using a fluorescence-activated cell sorter (FACS). Reactions inside individual liposomes can be measured in a high-throughput format provided that the encapsulated proteins give rise to a fluorescent signal such as by exhibiting fluorescence themselves or by catalyzing production of a fluorescent compound. The strategy of volume estimation was applied to in vitro selection experiments. The green fluorescent protein (GFP) gene was encapsulated into liposomes together with an in vitro translation system. Here liposomes carrying a single copy of the gene were identified using the internal aqueous volume information of individual liposomes, and those exhibiting higher green fluorescence intensity were sorted by the FACS machine. This system was able to enrich those encoding GFP with higher fluorescence intensity over those with lower intensity. These results suggest the possibility of performing evolutionary experiments in an environment that mimics the cell.