A Double-Strand Break Repair Component Is Essential for S Phase Completion in Fission Yeast Cell Cycling

Fission yeast rad22(+), a homologue of budding yeast RAD52, encodes a double-strand break repair component, which is dispensable for proliferation. We, however, have recently obtained a cell division cycle mutant with a temperature-sensitive allele of rad22(+), designated rad22-H6, which resulted from a point mutation in the conserved coding sequence leading to one amino acid alteration. We have subsequently isolated rad22(+) and its novel homologue rti1(+) as multicopy suppressors of this mutant. rti1(+) suppresses all the defects of cells lacking rad22(+). Mating type switch-inactive heterothallic cells lacking either rad22(+) or rti1(+) are viable, but those lacking both genes are inviable and arrest proliferation with a cell division cycle phenotype. At the nonpermissive temperature, a synchronous culture of rad22-H6 cells performs DNA synthesis without delay and arrests with chromosomes seemingly intact and replication completed and with a high level of tyrosine-phosphorylated Cdc2. However, rad22-H6 cells show a typical S phase arrest phenotype if combined with the rad1-1 checkpoint mutation. rad22(+) genetically interacts with rad11(+), which encodes the large subunit of replication protein A. Deletion of rad22(+)/rti1(+) or the presence of rad22-H6 mutation decreases the restriction temperature of rad11-A1 cells by 4-6 degrees C and leads to cell cycle arrest with chromosomes incompletely replicated. Thus, in fission yeast a double-strand break repair component is required for a certain step of chromosome replication unlinked to repair, partly via interacting with replication protein A.     

Functional connections between auditory cortical fields in humans revealed by Granger causality analysis of intra-cranial evoked potentials to sounds: comparison of two methods

Knowledge of neural interactions amongst cortical sites is important for understanding higher brain function. We studied such interactions using Granger causality (GC) to analyze auditory event-related potentials (ERPs) recorded directly and simultaneously from two physiologically identified and functionally interconnected auditory areas of cerebral cortex in human neurosurgical patients. Two methods of GC analysis were used and the results compared. Both approaches involved adaptive autoregressive modeling but differed from each other in other ways. Results obtained by using the two methods also differed. Fewer false-positive results were obtained using the method that suppressed the ERP non-stationarity and that expressed the GC as the sum of model coefficients, which suggests that this is the more appropriate approach for analyzing ERPs recorded directly from the human cortex.     

Mu Opioid Receptor Binding Correlates with Nicotine Dependence and Reward in Smokers

The rewarding effects of nicotine are associated with activation of nicotine receptors. However, there is increasing evidence that the endogenous opioid system is involved in nicotine''s rewarding effects. We employed PET imaging with [¹¹C]carfentanil to test the hypotheses that acute cigarette smoking increases release of endogenous opioids in the human brain and that smokers have an upregulation of mu opioid receptors (MORs) when compared to nonsmokers. We found no significant changes in binding potential (BP_ND) of [¹¹C]carfentanil between the placebo and the active cigarette sessions, nor did we observe differences in MOR binding between smokers and nonsmokers. Interestingly, we showed that in smokers MOR availability in bilateral superior temporal cortices during the placebo condition was negatively correlated with scores on the Fagerström Test for Nicotine Dependence (FTND). Also in smokers, smoking-induced decreases in [¹¹C]carfentanil binding in frontal cortical regions were associated with self-reports of cigarette liking and wanting. Although we did not show differences between smokers and nonsmokers, the negative correlation with FTND corroborates the role of MORs in superior temporal cortices in nicotine addiction and provides preliminary evidence of a role of endogenous opioid signaling in frontal cortex in nicotine reward.     

Network analysis of potential migration routes for Oriental White Storks (<Emphasis Type="Italic">Ciconia boyciana</Emphasis>)

From 1998 through to 2000, we satellite-tracked the movements of 13 Oriental White Storks (Ciconia boyciana) on their autumnal migration in order to identify their important stopover sites for preserving links from the Russian Far East breeding sites to the wintering sites in south-eastern China. New analytical methods of satellite tracking data were employed to derive robust information on the locations of stay sites, the number of stopovers made during migration, and the distance traveled without making stopovers. Based on the derived information, we modeled a stay site network as an abstraction of the storks potential migration routes from their breeding sites to wintering sites. Using network analysis techniques, we explored how the loss of stopover sites could affect the connectivity of potential migration routes. The results suggested that if the seashore stopover sites facing Bohai Bay in eastern China were lost, the storks wintering sites along the Yangtze River in south-eastern China would be isolated. Among the seashore stopover sites, Jiantuozhi Gley Mire (39.185°N, 118.627°E), located on the northern seashore of Bohai Bay, was considered particularly important for migrating storks, because it was used every year by the storks we tracked. If conservation needs of this critically located site fail to be addressed, the stay site network of storks can create weak links in the chain of migration and, if broken, storks will have great difficulties in completing their autumnal migration.     

Characterization of cancer stem cell properties of CD24 and CD26-positive human malignant mesothelioma cells

Malignant mesothelioma (MM) is an asbestos-related malignancy characterized by rapid growth and poor prognosis. In our previous study, we have demonstrated that several cancer stem cell (CSC) markers correlated with CSC properties in MM cells. Among these markers, we focused on two: CD24, the common CSC marker, and CD26, the additional CSC marker. We further analyzed the CSC properties of CD24 and CD26-positve MM cells. We established RNAi-knockdown cells and found that these markers were significantly correlated with chemoresistance, proliferation, and invasion potentials in vitro. Interestingly, while Meso-1 cells expressed both CD24 and CD26, the presence of each of these two markers was correlated with different CSC property. In addition, downstream signaling of these markers was explored by microarray analysis, which revealed that their expressions were correlated with several cancer-related genes. Furthermore, phosphorylation of ERK by EGF stimulation was significantly affected by the expression of CD26, but not CD24. These results suggest that CD24 and CD26 differentially regulate the CSC potentials of MM and could be promising targets for CSC-oriented therapy.     

Expression of microRNA miR-122 facilitates an efficient replication in nonhepatic cells upon infection with hepatitis C virus

Hepatitis C virus (HCV) is one of the most common etiologic agents of chronic liver diseases, including liver cirrhosis and hepatocellular carcinoma. In addition, HCV infection is often associated with extrahepatic manifestations (EHM), including mixed cryoglobulinemia and non-Hodgkin's lymphoma. However, the mechanisms of cell tropism of HCV and HCV-induced EHM remain elusive, because in vitro propagation of HCV has been limited in the combination of cell culture-adapted HCV (HCVcc) and several hepatic cell lines. Recently, a liver-specific microRNA called miR-122 was shown to facilitate the efficient propagation of HCVcc in several hepatic cell lines. In this study, we evaluated the importance of miR-122 on the replication of HCV in nonhepatic cells. Among the nonhepatic cell lines expressing functional HCV entry receptors, Hec1B cells derived from human uterus exhibited a low level of replication of the HCV genome upon infection with HCVcc. Exogenous expression of miR-122 in several cells facilitates efficient viral replication but not production of infectious particles, probably due to the lack of hepatocytic lipid metabolism. Furthermore, expression of mutant miR-122 carrying a substitution in a seed domain was required for efficient replication of mutant HCVcc carrying complementary substitutions in miR-122-binding sites, suggesting that specific interaction between miR-122 and HCV RNA is essential for the enhancement of viral replication. In conclusion, although miR-122 facilitates efficient viral replication in nonhepatic cells, factors other than miR-122, which are most likely specific to hepatocytes, are required for HCV assembly.     

Synergistic effects of primates and dung beetles on soil seed accumulation in snow regions

This study aimed to reveal the soil seed accumulation processes for endozoochorous plants in the heavy-snowfall forests of Japan, where seed dispersal agents are few when compared to tropical forests. We assessed (1) primary seed dispersal by Japanese macaques (Macaca fuscata) by identifying dispersed seeds found in their feces, and (2) secondary seed dispersal by dung beetles by using beads (as seeds mimics) of different sizes, to quantify the frequency of seed burial and burial depths. We studied this diplochorous system in different forest types (undisturbed beech forest, conifer plantation, and secondary beech-oak forest) and during the spring and summer seasons. The key findings were as follows: (1) macaques dispersed the seeds of 11 and 14 plant species during spring and summer, respectively; (2) seeds dispersed by macaques in the spring were smaller and twice as abundant than those dispersed in the summer; (3) although no differences were observed in the amount of beads buried by beetles between seasons, all bead sizes tended to be buried in deeper soil layers in the spring than in the summer; and (4) the seed supply to the soil in undisturbed beech forest and conifer plantation was greater than the one in secondary beech-oak forest. Similar to what has been observed in tropical forests, seeds defecated by frugivorous mammals can be successfully incorporated into the underground soil seed bank through a diplochorous macaque-beetle system in temperate forests of deep snow regions.     

Axin and the Axin/Arrow-binding protein DCAP mediate glucose-glycogen metabolism

Axin was found as a negative regulator of the canonical Wnt pathway. Human LRP5 was originally found as a candidate gene of insulin dependent diabetes mellitus (IDDM), but its Drosophila homolog, Arrow, works as a co-receptor of the canonical Wnt signal. In our previous paper, we found a new Drosophila Axin (Daxin)-binding SH3 protein, DCAP, a homolog of mammalian CAV family protein. Among the subtypes, DCAPL3 shows significant homology with CAP, an essential component of glucose transport in insulin signal. Further binding assay revealed that DCAP binds to not only Axin but also Arrow, and Axin binds to not only GSK3beta but also Arrow. However, overexpression and RNAi experiments of DCAP do not affect the canonical Wnt pathway. As DCAP is expressed predominantly in insulin-target organs, and as RNAi of DCAP disrupts the pattern of endogenous glycogen accumulation in late stage embryos, we suggest that DCAP is also involved in glucose transport. Moreover, early stage embryos lacking maternal Axin show significant delay of initial glycogen decomposition, and RNAi of Axin in S2 cells revealed quite increase of endogenous glycogen level as well as GSK3beta. These results suggest that Axin and DCAP mediate glucose-glycogen metabolism in embryo. In addition, the interaction among Axin, Arrow, and DCAP implies a possible cross-talk between Wnt signal and insulin signal.     

Torque-speed relationships of Na+-driven chimeric flagellar motors in Escherichia coli

The bacterial flagellar motor is a rotary motor in the cell envelope of bacteria that couples ion flow across the cytoplasmic membrane to torque generation by independent stators anchored to the cell wall. The recent observation of stepwise rotation of a Na⁺-driven chimeric motor in Escherichia coli promises to reveal the mechanism of the motor in unprecedented detail. We measured torque-speed relationships of this chimeric motor using back focal plane interferometry of polystyrene beads attached to flagellar filaments in the presence of high sodium-motive force (85 mM Na⁺). With full expression of stator proteins the torque-speed curve had the same shape as those of wild-type E. coli and Vibrio alginolyticus motors: the torque is approximately constant (at ∼ 2200 pN nm) from stall up to a “knee” speed of ∼ 420 Hz, and then falls linearly with speed, extrapolating to zero torque at ∼ 910 Hz. Motors containing one to five stators generated ∼ 200 pN nm per stator at speeds up to ∼ 100 Hz/stator; the knee speed in 4- and 5-stator motors is not significantly slower than in the fully induced motor. This is consistent with the hypothesis that the absolute torque depends on stator number, but the speed dependence does not. In motors with point mutations in either of two critical conserved charged residues in the cytoplasmic domain of PomA, R88A and R232E, the zero-torque speed was reduced to ∼ 400 Hz. The torque at low speed was unchanged by mutation R88A but was reduced to ∼ 1500 pN nm by R232E. These results, interpreted using a simple kinetic model, indicate that the basic mechanism of torque generation is the same regardless of stator type and coupling ion and that the electrostatic interaction between stator and rotor proteins is related to the torque-speed relationship.