Cdc7 kinase stimulates Aurora B kinase in M-phase.

The conserved serine-threonine kinase, Cdc7, plays a crucial role in initiation of DNA replication by facilitating the assembly of an initiation complex. Cdc7 is expressed at a high level and exhibits significant kinase activity not only during S-phase but also during G2/M-phases. A conserved mitotic kinase, Aurora B, is activated during M-phase by association with INCENP, forming the chromosome passenger complex with Borealin and Survivin. We show that Cdc7 phosphorylates and stimulates Aurora B kinase activity in vitro. We identified threonine-236 as a critical phosphorylation site on Aurora B that could be a target of Cdc7 or could be an autophosphorylation site stimulated by Cdc7-mediated phosphorylation elsewhere. We found that threonines at both 232 (that has been identified as an autophosphorylation site) and 236 are essential for the kinase activity of Aurora B. Cdc7 down regulation or inhibition reduced Aurora B activity in vivo and led to retarded M-phase progression. SAC imposed by paclitaxel was dramatically reversed by Cdc7 inhibition, similar to the effect of Aurora B inhibition under the similar situation. Our data show that Cdc7 contributes to M-phase progression and to spindle assembly checkpoint most likely through Aurora B activation.

Functional significance of the electrocorticographic auditory responses in the premotor cortex.

Other than well-known motor activities in the precentral gyrus, functional magnetic resonance imaging (fMRI) studies have found that the ventral part of the precentral gyrus is activated in response to linguistic auditory stimuli. It has been proposed that the premotor cortex in the precentral gyrus is responsible for the comprehension of speech, but the precise function of this area is still debated because patients with frontal lesions that include the precentral gyrus do not exhibit disturbances in speech comprehension. We report on a patient who underwent resection of the tumor in the precentral gyrus with electrocorticographic recordings while she performed the verb generation task during awake brain craniotomy. Consistent with previous fMRI studies, high-gamma band auditory activity was observed in the precentral gyrus. Due to the location of the tumor, the patient underwent resection of the auditory responsive precentral area which resulted in the post-operative expression of a characteristic articulatory disturbance known as apraxia of speech (AOS). The language function of the patient was otherwise preserved and she exhibited intact comprehension of both spoken and written language. The present findings demonstrated that a lesion restricted to the ventral precentral gyrus is sufficient for the expression of AOS and suggest that the auditory-responsive area plays an important role in the execution of fluent speech rather than the comprehension of speech. These findings also confirm that the function of the premotor area is predominantly motor in nature and its sensory responses is more consistent with the "sensory theory of speech production," in which it was proposed that sensory representations are used to guide motor-articulatory processes.

Mechanism of cancer cell death induced by depletion of an essential replication regulator.

BACKGROUND: Depletion of replication factors often causes cell death in cancer cells. Depletion of Cdc7, a kinase essential for initiation of DNA replication, induces cancer cell death regardless of its p53 status, but the precise pathways of cell death induction have not been characterized. METHODOLOGY/PRINCIPAL FINDINGS: We have used the recently-developed cell cycle indicator, Fucci, to precisely characterize the cell death process induced by Cdc7 depletion. We have also generated and utilized similar fluorescent cell cycle indicators using fusion with other cell cycle regulators to analyze modes of cell death in live cells in both p53-positive and -negative backgrounds. We show that distinct cell-cycle responses are induced in p53-positive and -negative cells by Cdc7 depletion. p53-negative cells predominantly arrest temporally in G2-phase, accumulating CyclinB1 and other mitotic regulators. Prolonged arrest at G2-phase and abrupt entry into aberrant M-phase in the presence of accumulated CyclinB1 are followed by cell death at the post-mitotic state. Abrogation of cytoplasmic CyclinB1 accumulation partially decreases cell death. The ATR-MK2 pathway is responsible for sequestration of CyclinB1 with 14-3-3σ protein. In contrast, p53-positive cancer cells do not accumulate CyclinB1, but appear to die mostly through entry into aberrant S-phase after Cdc7 depletion. The combination of Cdc7 inhibition with known anti-cancer agents significantly stimulates cell death effects in cancer cells in a genotype-dependent manner, providing a strategic basis for future combination therapies. CONCLUSIONS: Our results show that the use of Fucci, and similar fluorescent cell cycle indicators, offers a convenient assay system with which to identify cell cycle events associated with cancer cell death. They also indicate genotype-specific cell death modes induced by deficient initiation of DNA replication in cancer cells and its potential exploitation for development of efficient cancer therapies.

Electric-field-induced changes in absorption and fluorescence of the green fluorescent protein chromophore in a PMMA film.

External electric field effects on absorption, fluorescence, and fluorescence decay of p-HBDI that is a model compound of the chromophore of GFP have been examined in a poly(methyl methacrylate) film. The electroabsorption spectrum is similar in shape to the first derivative of the absorption spectrum, which results from the difference in molecular polarizability between the ground state and the Franck-Condon excited state. The electrophotoluminescence spectrum is dominated by the corresponding fluorescence spectrum, indicating the enhancement of the fluorescence intensity in the presence of external electric fields. The direct measurements of the electric field effect on the fluorescence decay profile suggest that the field-induced deceleration of the nonradiative process contributes to the increase in the fluorescence intensity in the presence of electric fields.

Tracing cadmium from culture to spikelet: noninvasive imaging and quantitative characterization of absorption, transport, and accumulation of cadmium in an intact rice plant.

We characterized the absorption and short-term translocation of cadmium (Cd) in rice (Oryza sativa 'Nipponbare') quantitatively using serial images observed with a positron-emitting tracer imaging system. We fed a positron-emitting 107Cd (half-life of 6.5 h) tracer to the hydroponic culture solution and noninvasively obtained serial images of Cd distribution in intact rice plants at the vegetative stage and at the grain-filling stage every 4 min for 36 h. The rates of absorption of Cd by the root were proportional to Cd concentrations in the culture solution within the tested range of 0.05 to 100 nm. It was estimated that the radial transport from the culture to the xylem in the root tissue was completed in less than 10 min. Cd moved up through the shoot organs with velocities of a few centimeters per hour at both stages, which was obviously slower than the bulk flow in the xylem. Finally, Cd arrived at the panicles 7 h after feeding and accumulated there constantly, although no Cd was observed in the leaf blades within the initial 36 h. The nodes exhibited the most intensive Cd accumulation in the shoot at both stages, and Cd transport from the basal nodes to crown root tips was observed at the vegetative stage. We conclude that the nodes are the central organ where xylem-to-phloem transfer takes place and play a pivotal role in the half-day travel of Cd from the soil to the grains at the grain-filling stage.

Photodynamic activity of C70 caged within surface-cross-linked liposomes.

[70]Fullerene (C(70)) encapsulated into a surface-cross-linked liposome, a so-called cerasome, was prepared by an exchange reaction incorporating C(70)gamma-cyclodextrin complexes into lipid membranes. Fullerene exchange in a cerasome-incorporated C(70) (CIC(70)), as well as in a lipid-membrane-incorporated C(70) (LMIC(70)), was completed within 1 min with stirring at 25 degrees C. CIC(70) was more resistant to lysis than LMIC(70) towards lysing agents such as surfactants. Furthermore, the photodynamic activity of CIC(70) in HeLa cells was similar to that of LMIC(70), indicating that C(70) can act as a photosensitizing drug (PS) without release from cerasome membranes. Thus, in contrast with general drug-delivery systems (DDSs), which require the drug to be released from the interior of liposomes, carriers for PSs for use in photodynamic therapy (PDT) do not necessarily need to release the drug. These results indicate that DDSs with high morphological stability can increase the residence time in blood and achieves tumor-selective drug delivery by the enhanced permeability and retention (EPR) effect.

Cdc7 as a potential new target for cancer therapy.

Cdc7 kinase plays crucial roles in firing of replication origins and in proper maintenance of replication forks, which are the sites of DNA replication. The inactivation of Cdc7 causes destabilization of replication forks leading to acute genomic instability and induces massive cell death preferentially in cancer cells. Thus, Cdc7 kinase may be a promising novel target for cancer therapy. Indeed, the first classes of Cdc7 inhibitors have been reported and have been shown to be effective in delaying tumor growth in animal models.

The autoregulation of nodulation mechanism is related to leaf development.

To understand the autoregulation of nodulation (AON) system, in which leguminous plants control the nodule number, we examined the details of the characteristics of hypernodulation soybean mutants NOD1-3 and NOD3-7. A microscopic study showed that NOD1-3 and NOD3-7 produced small-size leaves due to the smaller number of leaf cells, compared with the Williams parent. These phenotypes were not affected by inoculation with bradyrhizobia or nitrate supply. The AON signaling might be related to the control system of leaf cell proliferation. This hypothesis was strongly supported by the finding that activation of AON in wild types by inoculation leads to an increase in the cell number of leaves.

Nuclear import of Epstein-Barr virus nuclear antigen 1 mediated by NPI-1 (Importin alpha5) is up- and down-regulated by phosphorylation of the nuclear localization signal for which Lys379 and Arg380 are essential.

Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1) is essential for replication of episomal EBV DNAs and maintenance of latency. Multifunctional EBNA-1 is phosphorylated, but the significance of EBNA-1 phosphorylation is not known. Here, we examined the effects on nuclear translocation of Ser phosphorylation of the EBNA-1 nuclear localization signal (NLS) sequence, 379Lys-Arg-Pro-Arg-Ser-Pro-Ser-Ser386. We found that Lys379Ala and Arg380Ala substitutions greatly reduced nuclear transport and steady-state levels of green fluorescent protein (GFP)-EBNA1, whereas Pro381Ala, Arg382Ala, Pro384Ala, and Glu378Ala substitutions did not. Microinjection of modified EBNA-1 NLS peptide-inserted proteins and NLS peptides cross-linked to bovine serum albumin (BSA) showed that Ala substitution for three NLS Ser residues reduced the efficiency of nuclear import. Similar microinjection analyses demonstrated that phosphorylation of Ser385 accelerated the rate of nuclear import, but phosphorylation of Ser383 and Ser386 reduced it. However, transfection analyses of GFP-EBNA1 mutants with the Ser-to-Ala substitution causing reduced nuclear import efficiency did not result in a decrease in the nuclear accumulation level of EBNA-1. The results suggest dynamic nuclear transport control of phosphorylated EBNA-1 proteins, although the nuclear localization level of EBNA-1 that binds to cellular chromosomes and chromatin seems unchanged. The karyopherin alpha NPI-1 (importin alpha5), a nuclear import adaptor, bound more strongly to Ser385-phosphorylated NLS than to any other phosphorylated or nonphosphorylated forms. Rch1 (importin alpha1) bound only weakly and Qip1 (importin alpha3) did not bind to the Ser385-phosphorylated NLS. These findings suggest that the amino-terminal 379Lys-Arg380 is essential for the EBNA-1 NLS and that Ser385 phosphorylation up-regulates nuclear transport efficiency of EBNA-1 by increasing its binding affinity to NPI-1, while phosphorylation of Ser386 and Ser383 down-regulates it.

Monoclonal antibodies against regions topologically surrounding the homodimeric beta-barrel interface of Epstein-Barr virus nuclear antigen-1.

Epstein-Barr virus (EBV) nuclear antigen-1 (EBNA-1) is essential for maintenance of EBV latency. Four mouse monoclonal antibodies (mAbs) against the part of the EBNA-1 sequence (amino acids 451-641) containing the domain that forms a homodimeric eight-stranded beta-barrel were generated and characterized, examined for immunocytochemical staining, immunoblotting and isoelectric focusing of EBNA-1 proteins, and used to examine interactions between EBNA-1 polypeptides by far-Western blot assays. Far-Western blot analyses using the mAbs suggest that both the beta-strand (aa 593-604) and alpha helix (aa 568-582) are essential for EBNA-1 dimerization, consistent with yeast two-hybrid studies of mutant EBNA-1 polypeptides. These mAbs should be useful for studies on the structure and function of EBNA-1 proteins.

Maintenance of serum immunoglobulin G antibodies to Epstein-Barr virus (EBV) nuclear antigen 2 in healthy individuals from different age groups in a Japanese population with a high childhood incidence of asymptomatic primary EBV infection.

Immunoglobulin G (IgG) antibodies to Epstein-Barr virus (EBV) nuclear antigens 2 and 1 (EBNA-2 and EBNA-1, respectively) were studied using sera from healthy individuals of a population with a high incidence of asymptomatic primary EBV infections during infancy or childhood in Japan. Two CHO-K1 cell lines expressing EBNA-2 and EBNA-1 were used for anticomplement and indirect immunofluorescence assays. The positivity rate for EBNA-2 IgG rose in the 1- to 2-year age group, increased and remained at a plateau ( approximately 45%) between 3 and 29 years of age (3- to 4-, 5- to 9-, 10- to 14-, and 15- to 29-year age groups), and then reached 98% by age 40 (>/== 40-year age group). Both seropositivity for EBNA-1 and seropositivity for EBNAs in Raji cells (EBNA/Raji) were detected in the 1- to 2-year age group, remained high, and finally reached 100% by age 40. The geometric mean titer (GMT) of EBNA-2 IgG reached a plateau in the 5- to 9- and 10- to 14-year-old groups and remained elevated in the older age groups (15 to 29 and >/== 40 years). The GMT of EBNA-1 IgGs increased to a plateau in the 1- to 2-year-old group and remained unchanged in the older age groups. The GMT of EBNA/Raji IgGs also reached a plateau in the 1- to 2-year-old group, remained level throughout the 3- to 14-year age groups, and decreased in the 15- to 29-year-olds. EBNA-2 IgGs emerged earlier than EBNA-1 IgGs in 8 of 10 patients with infectious mononucleosis, who were between 1 and 27 years old, and declined with time in three of eight cases. These results suggest that EBNA-2 IgG antibodies evoked in young children by asymptomatic primary EBV infections remain elevated throughout life, probably because of reactivation of latent and/or exogenous EBV superinfection.

Epstein-Barr virus (EBV) nuclear antigen 1 colocalizes with cellular replication foci in the absence of EBV plasmids.

Epstein-Barr virus (EBV) EBNA-1 is the only EBV-encoded protein that is essential for the once-per-cell-cycle replication and maintenance of EBV plasmids in latently infected cells. EBNA-1 binds to the oriP region of latent EBV plasmids and cellular metaphase chromosomes. In the absence of oriP-containing plasmids, EBNA-1 was highly colocalized with cellular DNA replication foci that were identified by immunostaining S-phase cells for proliferating cell nuclear antigen and replication protein A (RP-A) in combination with DNA short pulse-labeling. For the association of EBNA-1 with the cellular replication focus areas, the EBNA-1 regions of amino acids (aa) 8 to 94 and/or aa 315 to 410, but not the RP-A-interacting carboxy-terminal region, were necessary. These results suggest a new aspect of latent virus-cell interactions.

Epstein-Barr virus nuclear antigen-1 is highly colocalized with interphase chromatin and its newly replicated regions in particular.

Epstein-Barr virus (EBV) nuclear antigen-1 (EBNA-1), which binds to both the EBV origin of replication (oriP) and metaphase chromosomes, is essential for the replication/retention and segregation/partition of oriP-containing plasmids. Here the chromosomal localization of EBNA-1 fused to green fluorescent protein (GFP-EBNA-1) is examined by confocal microscopy combined with a 'premature chromosome condensation' (PCC) procedure. Analyses show that GFP-EBNA-1 expressed in living cells that lack oriP plasmids is associated with cellular chromatin that has been condensed rapidly by the PCC procedure into identifiable forms that are unique to each phase of interphase as well as metaphase chromosomes. Studies of cellular chromosomal DNAs labelled with BrdU or Cy3-dUTP indicate that GFP-EBNA-1 colocalizes highly with the labelled, newly replicated regions of interphase chromatin in cells. These results suggest that EBNA-1 is associated not only with cellular metaphase chromosomes but also with condensing chromatin/chromosomes and probably with interphase chromatin, especially with its newly replicated regions.