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This paper proposes a general-purpose anomaly detection mechanism for Internet backbone traffic named GAMPAL (General-purpose Anomaly detection Mechanism using Prefix Aggregate without Labeled data). GAMPAL does not require labeled data to achieve general-purpose anomaly detection. For scalability to the number of entries in the BGP RIB (Border Gateway Protocol Routing Information Base), GAMPAL introduces prefix aggregate. The BGP RIB entries are classified into prefix aggregates, each of which is identified with the first three AS (Autonomous System) numbers in the AS_PATH attribute. GAMPAL establishes a prediction model for traffic sizes based on past traffic sizes. It adopts a LSTM-RNN (Long Short-Term Memory Recurrent Neural Network) model that focuses on the periodicity of the Internet traffic patterns at a weekly scale. The validity of GAMPAL is evaluated using real traffic information, BGP RIBs exported from the WIDE backbone network (AS2500), a nationwide backbone network for research and educational organizations in Japan, and the dataset of an ISP (Internet Service Provider) in Spain. As a result, GAMPAL successfully detects anomalies such as increased traffic due to an event, DDoS (Distributed Denial of Service) attacks targeted at a stub organization, a connection failure, an SSH (Secure Shell) scan attack, and anomaly spam. [ABSTRACT FROM AUTHOR]

Replacement of conventional Zircaloy fuel cladding with silicon carbide (SiC) fuel cladding is expected to significantly decrease the amount of hydrogen generated from fuel claddings by the reaction with steam during severe accidents. One of their critical issues addressed regarding practical application has been hydrothermal corrosion. Thus, the corrosion resistant coating technology using a Ti-Cr multilayer was developed to suppress silica dissolution from SiC fuel cladding into reactor coolant under normal operation. The effect of radiation on adhesion of the coating to SiC substrate and its microstructure characteristics were investigated following Si ion irradiation at 573 K up to 3 dpa for SiC. Measurement of swelling in pure Ti, pure Cr and SiC revealed that the maximum inner stress attributed to the swelling difference was generated between the coating and SiC substrate by irradiation of 1 dpa. No delamination and cracking were observed in cross-sectional specimens of the coated SiC irradiated up to 3 dpa. According to analyses using transmission electron microscopy, large void formation and cascade mixing due to irradiation were not observed in the coating. The swelling in the coating at 573 K was presumed to be caused by another mechanism during radiation such as point defects rather than void formation. [ABSTRACT FROM AUTHOR]

The circadian clock of cyanobacteria consists of only three clock proteins, KaiA, KaiB, and KaiC, which generate a circadian rhythm of KaiC phosphorylation in vitro. The adenosine triphosphatase (ATPase) activity of KaiC is the source of the 24-h period and temperature compensation. Although numerous circadian mutants of KaiC have been identified, the tuning mechanism of the 24-h period remains unclear. Here, we show that the circadian period of in vitro phosphorylation rhythm of mutants at position 402 of KaiC changed dramatically, from 15 h (0.6 d) to 158 h (6.6 d). The ATPase activities of mutants at position 402 of KaiC, without KaiA and KaiB, correlated with the frequencies (1/period), indicating that KaiC structure was the source of extra period change. Despite the wide-range tunability, temperature compensation of both the circadian period and the KaiC ATPase activity of mutants at position 402 of KaiC were nearly intact. We also found that in vivo and in vitro circadian periods and the KaiC ATPase activity of mutants at position 402 of KaiC showed a correlation with the side-chain volume of the amino acid at position 402 of KaiC. Our results indicate that residue 402 is a key position of determining the circadian period of cyanobacteria, and it is possible to dramatically alter the period of KaiC while maintaining temperature compensation. [ABSTRACT FROM AUTHOR]

KaiC, the core oscillator of the cyanobacterial circadian clock, is composed of an N-terminal C1 domain and a C-terminal C2 domain, and assembles into a double-ring hexamer upon ATP binding. Cyclic phosphorylation and dephosphorylation at Ser431 and Thr432 in the C2 domain proceed with a period of approximately 24 h in the presence of other clock proteins, KaiA and KaiB, but recent studies have revealed a crucial role for the C1 ring in determining the cycle period. In this study, we mapped dynamic structural changes of the C1 ring in solution using a combination of site-directed tryptophan mutagenesis and fluorescence spectroscopy. We found that the C1 ring undergoes a structural transition, coupled with ATPase activity and the phosphorylation state, while maintaining its hexameric ring structure. This transition triggered by ATP hydrolysis in the C1 ring in specific phosphorylation states is a necessary event for recruitment of KaiB, limiting the overall rate of slow complex formation. Our results provide structural and kinetic insights into the C1-ring rearrangements governing the slow dynamics of the cyanobacterial circadian clock. [ABSTRACT FROM AUTHOR]

The cyanobacterial circadian oscillator can be reconstituted in vitro. In the presence of KaiA and KaiB, the phosphorylation state of KaiC oscillates with a periodicity of ∼24 h. KaiC is a hexameric P-loop ATPase with autophosphorylation and autodephosphorylation activities. Recently, we found that dephosphorylation of KaiC occurs via reversal of the phosphorylation reaction: a phosphate group attached to Ser431/Thr432 is transferred to KaiC-bound ADP to generate ATP, which is subsequently hydrolyzed. This unusual reaction mechanism suggests that the KaiC phosphorylation rhythm is sustained by periodic shifts in the equilibrium of the reversible autophosphorylation reaction, the molecular basis of which has never been elucidated. Because KaiC-bound ATP and ADP serve as substrates for the forward and reverse reactions, respectively, we investigated the regulation of the nucleotide-bound state of KaiC. In the absence of KaiA, the condition in which the reverse reaction proceeds, KaiC favored the ADP-bound state. KaiA increased the ratio of ATP to total KaiC-bound nucleotides by facilitating the release of bound ADP and the incorporation of exogenous ATP, allowing the forward reaction to proceed. When both KaiA and KaiB were present, the ratio of ATP to total bound nucleotides exhibited a circadian rhythm, whose phase was advanced by several hours relative to that of the phosphorylation rhythm. Based on these findings, we propose that the direction of the reversible autophosphorylation reaction is regulated by KaiA and KaiB at the level of substrate availability and that this regulation sustains the oscillation of the phosphorylation state of KaiC. [ABSTRACT FROM AUTHOR]

In the cyanobacterium Synechococcus elongatus PCC 7942, the circadian clock entrains to a daily light/dark cycle. The transcription factor Pex is abundant under dark conditions and represses kaiA transcription to fine-tune the KaiC-based core circadian oscillator. The transcription of pex also increases during exposure to darkness; however, its mechanism is unknown. We performed a molecular genetic study by constructing a pex expression bioluminescent reporter and screening for brightly luminescent mutants by random insertion of a drug resistance gene cassette in the reporter genome. One mutant contained an insertion of an antibiotic resistance cassette in the cmpR locus, a transcriptional regulator of inorganic carbon concentration. Insertions of the cassette in the remaining two mutant genomes were in the genes encoding flavodoxin and a putative partner of an ABC transporter with unknown function (ycf22). We further analyzed the cmpR mutant to examine whether CmpR directly or indirectly targeted pex expression. In the cmpR mutant, the pex mRNA level was 1.8-fold that of the wild type, and its circadian peak phase in bioluminescence rhythm occurred 5 h later. Moreover, a high-light stress phenotype was present in the colony. The abnormalities were complemented by ectopic induction of the native gene. However, the cmpR/pex double mutation partly suppressed the phase abnormality (2.5 h). In vitro DNA binding analysis of CmpR showed positive binding to the psbAII promoter, but not to any pex DNA. We postulate that the phenotypes of cmpR-deficient cells were attributable mainly to a feeble metabolic and/or redox status. [ABSTRACT FROM AUTHOR]

Abstract: In the cyanobacterium Synechococcus elongatus, LabA negatively regulates circadian gene expression under the control of Kai-protein-based clock. Here we conducted a molecular genetic analysis of lalA, a paralog of labA. Although a lalA loss of function mutant did not exhibit any apparent phenotype under our experimental conditions, lalA overexpression inhibited cell growth and decreased cell viability. Moderate lalA overexpression brought about abnormalities in circadian gene expression: reduced amplitude of kaiBC expression rhythm, and altered peak and trough timing of psbAI and kaiA expression rhythms. These results imply that lalA is capable of affecting circadian gene expression and cell growth. [Copyright &y& Elsevier]

The circadian clock in cyanobacteria persists even without the transcription/translation feedbacks proposed for eukaryotic systems. The period of the cyanobacterial clock is tuned to the circadian range by the ATPase activity of a clock protein known as KaiC. Here, we provide structural evidence on how KaiC ticks away 24 h while coupling the ATPase activity in its N-terminal ring to the phosphorylation state in its C-terminal ring. During the phosphorylation cycle, the C-terminal domains of KaiC are repositioned in a stepwise manner to affect global expansion and contraction motions of the C-terminal ring. Arg393 of KaiC has a critical function in expanding the C-terminal ring and its replacement with Cys affects the temperature compensation of the period-a fundamental property of circadian clocks. The conformational ticking of KaiC observed here in solution serves as a timing cue for assembly/disassembly of other clock proteins (KaiA and KaiB), and is interlocked with its auto-inhibitory ATPase underlying circadian periodicity of cyanobacteria. [ABSTRACT FROM AUTHOR]

Abstract: Biochemical circadian oscillation of KaiC phosphorylation, by mixing three Kai proteins and ATP, has been proven to be the central oscillator of the cyanobacterial circadian clock. In vivo, the intracellular levels of KaiB and KaiC oscillate in a circadian fashion. By scrutinizing KaiC phosphorylation rhythm in a wide range of Kai protein concentrations, KaiA and KaiB were found to be “parameter-tuning” and “state-switching” regulators of KaiC phosphorylation rhythm, respectively. Our results also suggest a possible entrainment mechanism of the cellular circadian clock with the circadian variation of intracellular levels of Kai proteins. [Copyright &y& Elsevier]

Circadian kaiBC expression in the cyanobacterium Synechococcus elongatus PCC 7942 is generated by temporal information transmission from the KaiABC-based circadian oscillator to RpaA, a putative transcriptional factor, via the SasA-dependent positive pathway and the LabA-dependent negative pathway which is responsible for feedback regulation of KaiC. However, the labA/sasA double mutant has a circadian kaiBC expression rhythm, suggesting that there is an additional circadian output pathway. Here we describe a third circadian output pathway, which is CikA-dependent. The cikA mutation attenuates KaiC overexpression-induced kaiBC repression and exacerbates the low-amplitude phenotype of the labA mutant, suggesting that cikA acts as a negative regulator of kaiBC expression independent of the LabA-dependent pathway. In the labA/sasA/cikA triple mutant, kaiBC promoter activity becomes almost arrhythmic, despite preservation of the circadian KaiC phosphorylation rhythm, suggesting that CikA largely accounts for the residual kaiBCexpression rhythm observed in the labAlsasA double mutant. These results also strongly suggest that transcriptional regulation in the labA/sasA/cikA triple mutant is insulated from the circadian signals of the KaiABC-based oscillator. Based on these observations, we propose a model in which temporal information from the KaiABC-based circadian oscillator is transmitted to gene expression through three separate output pathways. [ABSTRACT FROM AUTHOR]

In the unicellular cyanobacterium Synechococcus elongatus PCC 7942. essentially all promoter activities are under the control of the circadian clock under continuous light (LL) conditions. Here, we used high-density oligonucleotide arrays to explore comprehensive profiles of genome-wide Synechococcus gene expression in wild-type, kaiABC-null, and kaiC-overexpressor strains under LI and continuous dark (DD) conditions. In the wild-type strains, >30% of transcripts oscillated significantly in a circadian fashion, peaking at subjective dawn and dusk. Such circadian control was severely attenuated in kaiABC-null strains. Although it has been proposed that KaiC globally represses gene expression, our analysis revealed that dawn-expressed genes were up-regulated by kaiC-overexpression so that the clock was arrested at subjective dawn. Transfer of cells to DD conditions from LL immediately suppressed expression of most of the genes, while the clock kept even time in the absence of transcriptional feedback. Thus, the Synechococcus genome seems to be primarily regulated by light! dark cycles and is dramatically modified by the protein-based circadian oscillator. [ABSTRACT FROM AUTHOR]

The three cyanobacterial Kai proteins and ATP are capable of generating an autonomous rhythm of KaiC phosphorylation in a test tube. As the period is ≈24 hours and is stable in a wide temperature range, this rhythm is thought to function as the basic oscillator of the cyanobacterial circadian system. We have examined the rhythm under various temperature cycles and found that it was stably entrained by a temperature cycle of 20-28 hours. As the period length was not altered by temperature, entrainment by period change could be excluded from possible mechanisms. Instead, temperature steps between 300 and 45°C and vice versa shifted the phase of the rhythm in a phase-dependent manner. Based on the phase response curves of the step-up and step-down in temperature, phase shift by single temperature pulse was estimated using a nonparametric entrainment model (discontinuous phase jump by external stimuli). The predicted phase shift was consistent with the experimentally measured phase shift. Next, successive phase shifts caused by repeated temperature cycles were computed by two phase response curves and compared with actual entrainment of the rhythm. As the entrainment pattern observed after various combinations of temperature cycles matched the prediction, it is likely that nonparametric entrainment functions even in the simple three-protein system. We also analyzed entrainment of KaiC phosphorylation by temperature cycle in cyanobacterial cells and found both the parametric and the nonparametric models function in vivo. [ABSTRACT FROM AUTHOR]

Circadian rhythms are found m organisms from cyanobacteria to plants and animals. In flowering plants, the circadian clock is involved in the regulation of various physiological phenomena, including growth, leaf movement, stomata opening, and floral transitions. Molecular mechanisms underlying the circadian clock have been identified using Arabidopsis (Arabidopsis thaliana); the functions and genetic networks of a number of clock-related genes, including CIRCADIAN CLOCK ASSOCI- ATEDI, LATE ELONGATED HYPOCOTYL (LHY), TIMING OF CAB EXPRESSIONI, GIGANTEA (GI), and EARLY FLOWER- ING3 (ELF3), have been analyzed. The degree to which clock systems are conserved among flowering plants, however, is still unclear. We previously isolated homologs for Arabidopsis clock-related genes from monocotyledon Lemna plants. Here, we report the physiological roles of these Lemna gibba genes (LgLHYHTI, LgLHYH2, LgGIH1, and LgELF3H1) in the circadian system. We studied the effects of overexpression and RNA interference (RNAi) of these genes on the rhythmic expression of morning- and evening-specific reporters. Overexpression of each gene disrupted the rhythmicity of either or both reporters, suggesting that these four homologs can be involved in the circadian system. RNAi of each of the genes except LgLHYH2 affected the bioluminescence rhythms of both reporters. These results indicated that these homologs are involved in the circadian system of Lemna plants and that the structure of the circadian clock is likely to be conserved between monocotyledons and dicotyledons. Interestingly, RNAi of LgGIH1 almost completely abolished the circadian rhythm; because this effect appeared to be much stronger than the phenotype observed in an Arabidopsis gi loss-of-function mutant, the precise role of each clock gene may have diverged in the clock systems of Lemna and Arabidopsis. [ABSTRACT FROM AUTHOR]

Singularity behaviour in circadian clocks — the loss of robust circadian rhythms following exposure to a stimulus such as a pulse of bright light — is one of the fundamental but mysterious properties of clocks. To quantitatively perturb and accurately measure the dynamics of cellular clocks, we synthetically produced photo-responsiveness within mammalian cells by exogenously introducing the photoreceptor melanopsin and continuously monitoring the effect of photo-perturbation on the state of cellular clocks. Here we report that a critical light pulse drives cellular clocks into singularity behaviour. Our theoretical analysis consistently predicts and subsequent single-cell level observation directly proves that desynchronization of individual cellular clocks underlies singularity behaviour. Our theoretical framework also explains why singularity behaviours have been experimentally observed in various organisms, and it suggests that desynchronization is a plausible mechanism for the observable singularity of circadian clocks. Importantly, these in vitro and in silico findings are further supported by in vivo observations that desynchronization underlies the multicell-level amplitude decrease in the rat suprachiasmatic nucleus induced by critical light pulses. [ABSTRACT FROM AUTHOR]

The cyanobacterial circadian oscillator can be reconstituted in vitro by mixing three purified clock proteins, KaiA, KaiB and KaiC, with ATP. The KaiC phosphorylation rhythm persists for at least 10 days without damping. By mixing oscillatory samples that have different phases and analyzing the dynamics of their phase relationships, we found that the robustness of the KaiC phosphorylation rhythm arises from the rapid synchronization of the phosphorylation state and reaction direction (phosphorylation or dephosphorylation) of KaiC proteins. We further demonstrate that synchronization is tightly linked with KaiC dephosphorylation and is mediated by monomer exchange between KaiC hexamers during the early dephosphorylation phase. This autonomous synchronization mechanism is probably the basis for the resilience of the cyanobacterial circadian system against quantitative fluctuations in clock components during cellular events such as cell growth and division. [ABSTRACT FROM AUTHOR]

Self-sustainable oscillation of KaiC phosphorylation has been reconstituted in vitro, demonstrating that this cycle is the basic time generator of the circadian clock of cyanobacteria. Here we show that the ATPase activity of KaiC satisfies the characteristics of the circadian oscillation, the period length, and the temperature compensation. KaiC possesses extremely weak but stable ATPase activity (15 molecules of ATP per day), and the addition of KaiA and KaiB makes the activity oscillate with a circadian period in vitro. The ATPase activity of KaiC is inherently temperature-invariant, suggesting that temperature compensation of the circadian period could be driven by this simple biochemical reaction. Moreover, the activities of wild-type KaiC and five period-mutant proteins are directly proportional to their in vivo circadian frequencies, indicating that the ATPase activity defines the circadian period. Thus, we propose that KaiC ATPase activity constitutes the most fundamental reaction underlying circadian periodicity in cyanobacteria. [ABSTRACT FROM AUTHOR]

The circadian phosphorylation cycle of the cyanobacterial clock protein KaiC has been reconstituted in vitro. The phosphorylation profiles of two phosphorylation sites in KaiC, serine 431 (S431) and threonine 432 (T432), revealed that the phosphorylation cycle contained four steps: (i) T432 phosphorylation; (ii) S431 phosphorylation to generate the double-phosphorylated form of KaiC; (iii) T432 dephosphorylation; and (iv) S431 dephosphorylation. We then examined the effects of mutations introduced at one KaiC phosphorylation site on the intact phosphorylation site. We found that the product of each step in the phosphorylation cycle regulated the reaction in the next step, and that double phosphorylation converted KaiC from an autokinase to an autophosphatase, whereas complete dephosphorylation had the opposite effect. These mechanisms serve as the basis for cyanobacterial circadian rhythm generation. We also found that associations among KaiA, KaiB, and KaiC result from S431 phosphorylation, and these interactions would maintain the amplitude of the rhythm. [ABSTRACT FROM AUTHOR]

KaiA, KaiB, and KaiC clock proteins from cyanobacteria and ATP are sufficient to reconstitute the KaiC phosphorylation rhythm in vitro, whereas almost all gene promoters are under the control of the circadian clock. The mechanism by which the KaiC phosphorylation cycle drives global transcription rhythms is unknown. Here, we report that RpaA, a potential DNA-binding protein that acts as a cognate response regulator of the KaiC-interacting kinase SasA, mediates between KaiC phosphorylation and global transcription rhythms. Circadian transcription was severely attenuated in sasA (Synechococcus adaptive sensor A)- and rpaA (regulator of phycobilisome-associated)-mutant cells, and the phosphotransfer activity from SasA to RpaA changed dramatically depending on the circadian state of a coexisting Kai protein complex in vitro. We propose a model in which the SasA-RpaA two-component system mediates time signals from the enzymatic oscillator to drive genome-wide transcription rhythms in cyanobacteria. Moreover, our results indicate the presence of secondary output pathways from the clock to transcription control, suggesting that multiple pathways ensure a genome-wide circadian system. [ABSTRACT FROM AUTHOR]

The Lemna genus is a group of monocotyledonous plants with tiny, floating bodies. Lemna gibba G3 and L. paucicostata 6746 were once intensively analyzed for physiological timing systems of photoperiodic flowering and circadian rhythms since they showed obligatory and sensitive photoperiodic responses of a long-day and a short-day plant, respectively. We attempted to approach the divergence of biological timing systems at the molecular level using these plants. We first employed molecular techniques to study their circadian clock systems. We developed a convenient bioluminescent reporter system to monitor the circadian rhythms of Lemna plants. As in Arabidopsis, the Arabidopsis CCA1 promoter produced circadian expression in Lemna plants, though the phases and the sustainability of bioluminescence rhythms were somewhat diverged between them. Lemna homologs of the Arabidopsis clock-related genes LHY/CCA1, GI, ELF3 and PRRs were then isolated as candidates for clock-related genes in these plants. These genes showed rhythmic expression profiles that were basically similar to those of Arabidopsis under light–dark conditions. Results from co-transfection assays using the bioluminescence reporter and overexpression effectors suggested that the LHY and GI homologs of Lemna can function in the circadian clock system like the counterparts of Arabidopsis. All these results suggested that the frame of the circadian clock appeared to be conserved not only between the two Lemna plants but also between monocotyledons and dicotyledons. However, divergence of gene numbers and expression profiles for LHY/CCA1 homologs were found between Lemna, rice and Arabidopsis, suggesting that some modification of clock-related components occurred through their evolution. [ABSTRACT FROM AUTHOR]

In the cyanobacterium, Synechococcus elongatus PCC 7942, the kaiBC operon is upregulated by the KaiA protein and downregulated by the KaiC protein to generate circadian oscillation. We investigated the regulation of kaiBC transcription. A primer extension and deletion analyses of the upstream region mapped the sufficient promoter region (SPR) to base pairs −55 to +1 (the transcription start site, TSS) and identified a constitutive negative regulatory region upstream of the SPR (base pairs −897 to −56) that extended into the coding sequence of kaiA. Base-pair substitution within the SPR identified a sequence from −52 to −28 that was the essential element for transcription. Most of the examined sequences drove rhythmic expression of a luxAB reporter that was similar to the expression driven by the kaiBC promoter (P kaiBC) and responded to the overexpression of kaiA or kaiC, even in a promoter activity range of 1–8000%. These results indicate that circadian feedback regulation by KaiA and KaiC is addressed to a global step preceding transcription driven by P kaiBC. However, increasing or decreasing the intrinsic activity of P kaiBC greatly affected the rhythm, suggesting that constitutive adjustment of P kaiBC activity by the sequences identified here is essential for the oscillator. [ABSTRACT FROM AUTHOR]

Cyanobacteria are the simplest organisms known to exhibit circadian rhythms and have provided experimental model systems for the dissection of basic properties of circadian organization at the molecular, physiological, and ecological levels. This review focuses on the molecular and genetic mechanisms of circadian rhythm generation in cyanobacteria. Recent analyses have revealed the existence of multiple feedback processes in the prokaryotic circadian system and have led to a novel molecular oscillator model. Here, the authors summarize current understanding of, and open questions about, the cyanobacterial oscillator. [ABSTRACT FROM AUTHOR]

In the cyanobacterium Synechococcus elongatus PCC 7942, KaiA, KaiB, and KaiC are essential proteins for the generation of a circadian rhythm. KaiC is proposed as a negative regulator of the circadian expression of all genes in the genome, and its phosphorylation is regulated positively by KaiA and negatively by KaiB and shows a circadian rhythm in vivo. To study the functions of KaiC phosphorylation in the circadian clock system, we identified two autophosphorylation sites, Ser-431 and Thr-432, by using mass spectrometry (MS). We generated Synechococcus mutants in which these residues were substituted for alanine by using site-directed mutagenesis. Phosphorylation of KaiC was reduced in the single mutants and was completely abolished in the double mutant, indicating that KaiC is also phosphorylated at these sites in vivo. These mutants lost circadian rhythm, indicating that phosphorylation at each of the two sites is essential for the control of the circadian oscillation. Although the nonphosphorylatable mutant KaiC was able to form a hexamer in vitro, it failed to form a clock protein complex with KaiA, KaiB, and SasA in the Synechococcus cells. When nonphosphorylatable KaiC was overexpressed, the kaiBC promoter activity was only transiently repressed. These results suggest that KaiC phosphorylation regulates its transcriptional repression activity by controlling its binding affinity for other clock proteins. [ABSTRACT FROM AUTHOR]

The high-conversion boiling water reactor (HCBWR) has been studied as one of the next-generation BWRs. The HCBWR can be improved by the use of island-type fuel, which has mixed-oxide rods in the bundle interior and uranium rods only in the small region of the periphery, to have inherently negative void coefficient (i.e., negative void coefficient in infinite lattice configuration). The proposed reactor concept also has the sustainability to extend the light water reactor's period by ∼ 180 yr and the compatibility with a conventional BWR system such that only substitution of fuel bundles and control rods is required. As an example case, the high-conversion advanced boiling water reactor II (ABWR-II) is evaluated. [ABSTRACT FROM AUTHOR]

A kalABC clock gene cluster was previously identified from cyanobacterium Synechococcus elongatus PCC 7942, and the feedback regulation of kai genes was proposed as the core mechanism generating circadian oscillation. In this study, we confirmed that the Kai-based oscillator is the dominant circadian oscillator functioning in cyanobacteria. We probed the nature of this regulation and found that excess KaiC represses not only kaiBC but also the rhythmic components of all genes in the genome. This result strongly suggests that the KaiC protein primarily coordinates genomewide gene expression, including its own expression. We also found that a promoter derived from E. coli is feedback controlled by KaiC and restores the complete circadian rhythm in kaiBC-inactivated arrhythmic mutants, provided it can express kaiB and kaiC genes at an appropriate level. Unlike eukaryotic models, specific regulation of the kaiBC promoter is not essential for cyanobacterial circadian oscillations. [ABSTRACT FROM AUTHOR]

Purpose. Rho-associated coiled-coil-forming protein kinase (ROCK) is pivotally involved in invasion by tumor cells and their evolution to metastasis. We have developed a novel inhibitor of ROCK, Wf-536 [(+)-(R)-4-(1-aminoethyl)-N-(4-pyridyl) benzamide monohydrochloride]. In the present study, we investigated its effect on in vitro invasion and in vivo pulmonary metastasis of B16 melanoma. Methods. The following were evaluated: the anti-invasive effect of Wf-536 against the motility of mouse B16BL6 melanoma cells through a culture insert layered with reconstituted basement membrane (Matrigel); the cytotoxic effect of Wf-536 in the same cell line; the antimetastatic effect of Wf-536, administered by osmotic pump, on spontaneous pulmonary metastasis following subcutaneous injection of B16BL6 melanoma in mice; and the inhibitory effect of orally administered Wf-536, alone or in combination with the antineoplastic drug paclitaxel, on pulmonary metastasis of intravenously injected B16F10 melanoma in mice. Results. Wf-536 inhibited in vitro invasion by B16BL6 cells significantly and in a concentration-dependent manner and displayed an anti-invasive effect under conditions of both chemotaxis and chemokinesis. No cytotoxic effect was observed at any of the concentrations used. In vivo, Wf-536 administration suppressed tumor colony formation on the lung surface in a dose-dependent manner (0.3–3 mg/kg per day), with a metastasis inhibition rate of 95% at 3 mg/kg per day. In experimental metastasis of B16F10 melanoma, oral administration of Wf-536 significantly decreased tumor colony formation in the lung, with an inhibition rate of 41% at 3 mg/kg per day. The inhibition rate of paclitaxel (5 mg/kg per day) was 27%. The combination of Wf-536 and paclitaxel produced a synergistic effect on B16F10 metastasis and a 68% inhibition rate. Wf-536 administration at the doses used did not alter body weight, blood pressure or the health of treated animals as compared to vehicle-treated controls. Conclusion. The results suggest that Wf-536 is a potentially valuable drug for preventing tumor metastasis both in monotherapy and in combination with an antineoplastic drug. [ABSTRACT FROM AUTHOR]

Summary 1. Rho-associated coiled-coil forming protein serine/threonine kinase (ROCK) is involved in the development of tumour metastasis. Wf-536, (+)-(R )-4-(1-Aminoethyl)-N -(4-pyridyl) benzamide monohydrochloride, a novel inhibitor of ROCK, inhibits tumour metastasis in some animal models. To metastasise, tumour cells have to disturb the tight intercellular junctions and the basement membrane matrix of the host tissue, which, respectively, create an intercellular barrier and the extracellular membrane. To clarify the mechanism of Wf-536 in inhibition of tumour metastasis, we analysed the effect of Wf-536 on the transition of tumour cells through the host cell layer and the basement membrane in in vitro systems. 2. In a coculture system of human fibrosarcoma HT1080 cells plated on a monolayer of human ECV304 cells, Wf-536 (0.3–3 µmol/L) inhibited the paracellular infiltration of tumour cells. 3. Wf-536 (3–30 µmol/L) inhibited the invasion of tumour cells through the reconstituted basement membrane (Matrigel) layer. 4. Wf-536 (10–30 µmol/L) inhibited the migration of tumour cells. At 0.3–3 µmol/L, Wf-536 also restrained hepatocyte growth factor/scatter factor (HGF)-induced increases in paracellular permeability of the ECV304 cell layer. 5. These results suggest that Wf-536 suppresses tumour metastasis by both enhancing the barrier function of host cell layers and inhibiting tumour cell motility at the stage of host tissue penetration by metastatic tumour cells. [ABSTRACT FROM AUTHOR]

Cyanobacterial clock proteins KaiA and KaiC are proposed as positive and negative regulators in the autoregulatory circadian kaiBC expression, respectively. Here, we show that activation of kaiBC expression by kaiA requires KaiC, suggesting a positive feedback control in the cyanobacterial clockwork. We found that robust circadian phosphorylation of KaiC. KaiA was essential for in vivo KaiC phosphorylation and activated in vitro KaiC autophosphorylation. These effects of KaiA were attenuated by the kaiA2 long period mutation. Both the long period phenotype and the abnormal KaiC phosphorylation in this mutant were suppressed by a previously undocumented kaiC mutation. We propose that KaiAstimulated circadian KaiC phosphorylation is important for circadian timing. [ABSTRACT FROM AUTHOR]

Circadian rhythms have been observed in innumerable physiological processes in most of organisms. Recent molecular and genetic studies on circadian clocks in many organisms have identified and characterized several molecular regulatory factors that contribute to generation of such rhythms. The cyanobacterium is the simplest organism known to harbor circadian clocks, and it has become one of most successful model organisms for circadian biology. In this review, we will briefly summarize physiological observations and consideration of circadian rhythms in cyanobacteria, molecular genetics of the clock using Synechococcus, and current knowledge of the input and output pathways that support the cellular circadian system. Finally, we will document some current problems in the studies on the cyanobacterial circadian clock. [ABSTRACT FROM PUBLISHER]

Focuses on the circadian clock in cyanobacteria. Temporal orchestration of gene expression in cyanobacteria; Adaptive significance of circadian clock in cyanobacteria; Synechococcus as a model system for molecular genetic dissection of the circadian oscillator.

The kai gene cluster, which is composed of three genes, kaiA, kaiB and kaiC, is essential for the generation of circadian rhythms in the unicellular cyanobacterium Synechococcus sp. strain PCC 7942. Here we demonstrate the direct association of KaiA, KaiB and KaiC in yeast cells using the two-hybrid system, in vitro and in cyanobacterial cells. KaiC enhanced KaiA­KaiB interaction in vitro and in yeast cells, suggesting that the three Kai proteins were able to form a heteromultimeric complex. We also found that a long period mutation kaiA1 dramatically enhanced KaiA­KaiB interaction in vitro. Thus, direct protein­protein association among the Kai proteins may be a critical process in the generation of circadian rhythms in cyanobacteria. [ABSTRACT FROM AUTHOR]