Replication initiator DnaA of Escherichia coli changes its assembly form on the replication origin during the cell cycle.

DnaA is a replication initiator protein that is conserved among bacteria. It plays a central role in the initiation of DNA replication. In order to monitor its behavior in living Escherichia coli cells, a nonessential portion of the protein was replaced by a fluorescent protein. Such a strain grew normally, and flow cytometry data suggested that the chimeric protein has no substantial loss of the initiator activity. The initiator was distributed all over the nucleoid. Furthermore, a majority of the cells exhibited certain distinct foci that emitted bright fluorescence. These foci colocalized with the replication origin (oriC) region and were brightest during the period spanning the initiation event. In cells that had undergone the initiation, the foci were enriched in less intense ones. In addition, a significant portion of the oriC regions at this cell cycle stage had no colocalized DnaA-enhanced yellow fluorescent protein (EYFP) focus point. It was difficult to distinguish the initiator titration locus (datA) from the oriC region. However, involvement of datA in the initiation control was suggested from the observation that, in DeltadatA cells, DnaA-EYFP maximally colocalized with the oriC region earlier in the cell cycle than it did in wild-type cells and oriC concentration was increased.

Initiator titration complex formed at datA with the aid of IHF regulates replication timing in Escherichia coli.

The initiation of replication in Escherichia coli is negatively controlled by a mechanism referred to as 'initiator titration', a process by which the initiator protein, DnaA, is titrated to newly replicated binding sequences on the chromosome to reduce the initiation potential for replication. Initiator titration occurs predominantly at the datA locus that binds exceptionally large amounts of DnaA molecules to prevent aberrant initiations. We found that this was enabled by integration host factor (IHF). Within datA, there is a consensus IHF recognition sequence between the two DnaA recognition sequences (DnaA boxes) essential for its function. Binding of IHF to this site was demonstrated both in vitro and in vivo. Disruption of the core sequence in the consensus of the IHF-binding resulted in increased origin concentration as observed in Delta datA cells. Furthermore, the number of DnaA molecules bound to datA was reduced in cells carrying a disruption in the IHF-binding core sequence. The IHF-binding site and the essential DnaA boxes had to be located at a proper distance and orientation to maintain the accurate initiation timing. Therefore, IHF is a unique element in the control of replication initiation that acts negatively at datA, while known to act as a positive regulator at oriC.

Determination of the minimum domain II size of Escherichia coli DnaA protein essential for cell viability.

The DnaA protein is the bacterial initiator of replication at a unique chromosomal site, oriC. It is present in all bacterial species and has a conserved structure with four domains. The structures of domains I and III-IV have been solved recently for some bacterial species, and the molecular process leading to the initiation event has been investigated in detail. On the other hand, domain II appears to have no rigid structure and is assumed to be a flexible linker connecting the N-terminal domain I and the C-terminal domains III-IV. It differs significantly in length and amino acid sequence among bacterial species. Whether or not domain II has any function(s) to initiate replication is unknown. The precise borders at both of its ends as well as its essential portions for cell viability are also unknown. In this study, we introduced systematic deletions into the domain II region on the chromosomal dnaA gene of Escherichia coli and examined their effect on cell physiology. Stretches of 30-36 consecutive amino acid residues could be deleted from various portions between the 78th and the 136th residues without affecting cell viability. We propose that domain II of E. coli DnaA is from the 79th to the 135th residues and at least 21-27 residues are required as a spacer to keep domains I and III-IV in the correct positions.

[Questionnaire survey of dentists who made their own mouthguards].

PURPOSE: Most of the mouthguards on the market are inferior in fit and occlusion related to feeling and injury prevention capacity. Therefore, it is necessary to use appropriate custom-made mouth-guards. This research aimed to obtain data for the selection, improvement, and spread of mouthguards in the future. A questionnaire survey of dentists who had made four kinds of mouthguard was conducted in a mouthguard seminar. METHODS: The questionnaire survey concerning "feeling", "difficulty of production", and "selection when considering use and spread" was done for four kinds of mouthguard. The evaluations were made using a ten-point method with the Kruskal-Wallis test and Mann-Whitney U-test. RESULTS: Concerning the feeling: The laminated mouthguard was evaluated the highest, followed in order by improvement type, vacuum, and boil & bite. Concerning the production: The evaluation differed from other questionnaire items. No significant difference was found among all four kinds of mouthguard, so there was no difference in the fabrication difficulty. Concerning the selection and spread: The evaluation was almost the same as for the feeling. The laminated mouthguard was assessed to be the best mouthguard. CONCLUSIONS: The boil & bite mouthguard which is widespread was evaluated the lowest in all items except production. Therefore, it is necessary to encourage players to use an appropriate custom-made type in view of safety, wearing feeling, and dental occlusion.

Suppressive effect of trehalose on acrylamide formation from asparagine and reducing saccharides.

The influence of saccharides on the formation of acrylamide (AcA) was investigated. The reducing saccharides reacted with asaparagine to form AcA, but the non-reducing saccharides, except sucrose, gave no AcA. AcA formation from a mixture containing glucose and asaparagaine was suppressed by the non-reducing saccharides, especially trehalose (76% suppression) and neotrehalose (75% suppression). Glucose is heat-degraded into pyruvaldehyde and 5-hydroxymethyl-2-furfural in the water system. The degradation products react with asparagines to generate AcA. Trehalose appears to inhibit not only the formation of these intermediates and asparagines for AcA, but also the AcA formation from these intermediates.

Are all mouthguards the same and safe to use? The influence of occlusal supporting mouthguards in decreasing bone distortion and fractures.

The safety benefits of mouthguards have been demonstrated in many studies, with many authors and sports dentists strongly recommending the wearing of mouthguards. However, wearing a mouthguard with incorrect occlusion might cause a variety of problems. It comes as no surprise that a traumatic blow to the chin, while wearing an insufficient mouthguard lacking anterior contact, can result in severe distortions to the mandibular bone, and bone fractures. The aim of this study was to clarify how ineffective insufficient occlusal supporting mouthguards are and how dangerous they can be to use. Consequently, in this study, occlusal supportive areas were varied and accelerations of head and distortions of the mandible were measured using an artificial skull model and a pendulum impact device. As a result, the distortions of the mandible tended to increase as the supported area decreased. On the contrary, accelerations of the head decreased as the occlusion part decreased. Thus, a lot of impact energy was consumed in the distortion of the mandible; accordingly, it seemed that only a little destructive energy was transferred to the head. From this study, it would seem that wearing a mouthguard, which is insufficient in the occlusion, has the potential of causing a bone fracture of the mandible. Consequently, mouthguards should have proper occlusion.

The datA locus predominantly contributes to the initiator titration mechanism in the control of replication initiation in Escherichia coli.

Replication of the Escherichia coli chromosome is initiated synchronously from all origins (oriC) present in a cell at a fixed time in the cell cycle under given steady state culture conditions. A mechanism to ensure the cyclic initiation events operates through the chromosomal site, datA, which titrates exceptionally large amounts of the bacterial initiator protein, DnaA, to prevent overinitiation. Deletion of the datA locus results in extra initiations and altered temporal control of replication. There are many other sites on the E. coli chromosome that can bind DnaA protein, but the contribution of these sites to the control of replication initiation has not been investigated. In the present study, seven major DnaA binding sites other than datA have been examined for their influence on the timing of replication initiation. Disruption of these seven major binding sites, either individually or together, had no effect on the timing of initiation of replication. Thus, datA seems to be a unique site that adjusts the balance between free and bound DnaA to ensure that there is only a single initiation event in each bacterial cell cycle. Mutation either in the second or the third DnaA box (a 9 basepair DnaA-binding sequence) in datA was enough to induce asynchronous and extra initiations of replication to a similar extent as that observed with the datA-deleted strain. These DnaA boxes may act as cores for the cooperative binding of DnaA to the entire datA region.