Tel2 is required for activation of the Mrc1-mediated replication checkpoint

Proteins belonging to the Tel2/Rad-5/Clk-2 family are conserved among eukaryotes and are involved in various cellular processes, such as cell proliferation, telomere maintenance, the biological clock, and the DNA damage checkpoint. However, the molecular mechanisms underlying the functions of these molecules remain largely unclear. Here we report that in the fission yeast, Schizosaccharomyces pombe, Tel2 is required for efficient phosphorylation of Mrc1, a mediator of DNA replication checkpoint signaling, and for activation of Cds1, a replication checkpoint kinase, when DNA replication is blocked by hydroxyurea. In fact, Tel2 is required for survival of replication fork arrest and for the replication checkpoint in cells lacking Chk1, another checkpoint kinase the role of which overlaps that of Cds1 in cell cycle arrest by replication block. In addition, Tel2 plays important roles in entry into S phase and in genome stability. Tel2 is essential for vegetative cell growth, and the tel2Delta strain accumulated cells with 1C DNA content after germination. In the absence of hydroxyurea, Tel2 is vital in the mutant lacking Swi1, a component of the replication fork protection complex, and multiple Rad22 DNA repair foci were frequently observed in Tel2-repressed swi1Delta cells especially at S phase. In contrast, the cds1Deltaswi1Delta mutant did not show such lethality. These results indicate that S. pombe Tel2 plays important roles in the Mrc1-mediated replication checkpoint as well as in the Cds1-independent regulation of genome integrity.     

Formation mechanism of 2,6-dimethyl-2,6-octadienes from thermal decomposition of linalyl beta-D-glucopyranoside

Thermally decomposed products of (+/-)-linalyl beta-D-glucoside were analyzed by GC and GC/MS. 2,6-dimethyl-2,6-octadienes produced by mild pyrolysis of linalyl beta-D-glucopyranoside under a vacuum were detected and characterized by MS and NMR spectroscopy. This suggests that 2,6-dimethyl-2,6-octadienes are produced during thermal decomposition of the glucoside via proton transfer from the anomeric position to C-6 in the aglycon moiety. A stable isotope labeling experiment directly indicated the new reaction mechanism.     

Mutant presenilin (A260V) affects Rab8 in PC12D cell

Most familial early-onset Alzheimer's disease (FAD) is caused by mutations in the presenilin-1 (PS1) gene. Abeta is derived from amyloid precursor protein (APP) and an increased concentration of Abeta 42 is widely believed to be a pathological hallmark of abnormal PS function. Therefore, the interaction between PS1 and APP is a central theme in attempts to clarify the molecular mechanism of AD. To examine the effect of PS1 mutations on APP metabolism, we made PC12D cell lines that express human PS1 or mutant PS1 (A260V). In PC12D cells expressing the PS1A260V mutant, we found that Rab8, a GTPase involved in transport from the trans-Golgi network (TGN) to the plasma membrane (PM), was significantly reduced in PC12D cells expressing the A260V mutant and that APP C-terminal fragment (CTF), the direct precursor of Abeta, accumulated in the heavy membrane fraction including membrane vesicles involved in TGN-to-PM transport. Furthermore, the total intracellular Abeta production was reduced in these cells. Combined together, we have observed that PS1 mutation disturbs membrane vesicle transport, resulting in prolonged residence of APP CTF during TGN-to-PM transport pathway. Therefore, it is highly likely that reduction of Abeta is closely related to the retention of APP CTF during TGN-to-PM transport.     

Cohesin defects lead to premature sister chromatid separation,kinetochore dysfunction,and spindle-assembly checkpoint activation

Scc1/Mcd1 is a component of the cohesin complex that plays an essential role in sister chromatid cohesion in eukaryote cells. Knockout experiments of this gene have been described in budding yeast, fission yeast, and chicken cells, but no study has been reported on human Scc1 thus far. In this study, we found that an N-terminally truncated human Scc1 shows a dominant-negative effect, and we examined the phenotypes of human cells defective in Scc1 function. Scc1 defects led to failure of sister chromatid cohesion in both interphase and mitotic cells. Interestingly, four chromatids derived from two homologues occupied four distinct territories in the nucleus in chromosome painting experiments. In mitotic Scc1-defective cells, chromatids were disjoined with normal condensation, and the spindle-assembly checkpoint was activated. We also found that, although the disjoined kinetochore (half-kinetochore) in Scc1-defective cells contains CENP-A, -B, -C, and -E normally, it apparently does not establish the kinetochore-microtubule association. These results indicate that Scc1 is essential for the association of kinetochores with microtubules.     

Usefulness of HMG-CoA reductase inhibitor in Japanese hyperlipidemic women within seven years of menopause

OBJECTIVE: To assess the therapeutic value of treatment with an HMG-CoA reductase inhibitor in women with hypoestrogenic hyperlipidemia caused by menopause. DESIGN: Fifty-six women with total cholesterol (TC) levels of 220 mg/dl or more who were within 7 years of menopause were randomly assigned to receive an HMG-CoA reductase inhibitor (pravastatin 10 mg/day; treated group, 26 patients) or no medical treatment (nontreated group, 30 patients) in this 6-month nonblinded prospective trial. RESULTS: In the treated group, the mean (SD) TC levels decreased significantly from 254.5+/-22.3 mg/dl at baseline to 204.7+/-22.2 mg/dl (19.6%), and the mean low-density lipoprotein cholesterol (LDL-C) level decreased significantly from 146.7+/-30.5 to 104.3+/-22.5 mg/dl (28.9%); the mean arteriosclerotic index decreased significantly from 2.98 to 2.08 (30.2%). There were no significant changes in either triglyceride levels or high-density lipoprotein cholesterol (HDL-C) levels. In the nontreated group, there were no significant changes in the TC, HDL-C, LDL-C, or triglyceride levels; there was also no change in the arteriosclerotic index. After 6 months, the TC level, LDL-C level, and arteriosclerotic index were significantly lower in the treated group compared with the nontreated group (p<0.01). CONCLUSIONS: The results indicate that the HMG-CoA reductase inhibitor lowered TC and LDL-C levels and was useful in the treatment of hypoestrogenic hyperlipidemia for periods of at least 6 months.     

Alterations of DNA and chromatin structures at telomeres and genetic instability in mouse cells defective in DNA polymerase alpha

Telomere length is controlled by a homeostatic mechanism that involves telomerase, telomere-associated proteins, and conventional replication machinery. Specifically, the coordinated actions of the lagging strand synthesis and telomerase have been argued. Although DNA polymerase alpha, an enzyme important for the lagging strand synthesis, has been indicated to function in telomere metabolism in yeasts and ciliates, it has not been characterized in higher eukaryotes. Here, we investigated the impact of compromised polymerase alpha activity on telomeres, using tsFT20 mouse mutant cells harboring a temperature-sensitive polymerase alpha mutant allele. When polymerase alpha was temperature-inducibly inactivated, we observed sequential events that included an initial extension of the G-tail followed by a marked increase in the overall telomere length occurring in telomerase-independent and -dependent manners, respectively. These alterations of telomeric DNA were accompanied by alterations of telomeric chromatin structures as revealed by quantitative chromatin immunoprecipitation and immunofluorescence analyses of TRF1 and POT1. Unexpectedly, polymerase alpha inhibition resulted in a significantly high incidence of Robertsonian chromosome fusions without noticeable increases in other types of chromosomal aberrations. These results indicate that although DNA polymerase alpha is essential for genome-wide DNA replication, hypomorphic activity leads to a rather specific spectrum of chromosomal abnormality.     

Effects of a 50 Hz electric field on plasma lipid peroxide level and antioxidant activity in rats

The effects of exposure to extremely low frequency electric fields (ELF EFs) on plasma lipid peroxide levels and antioxidant activity (AOA) in Sprague-Dawley rats were studied. The test was based on comparisons among rats treated with a combination of the oxidizing agent, 2,2'-azobis(2-aminopropane) dihydrochloride (AAPH) and 50 Hz EF of 17.5 kV/m intensity for 15 min per day for 7 days, AAPH alone, EF alone or no treatment. EF significantly decreased the plasma peroxide level in rats treated with AAPH, similar to treatment by ascorbic acid or the superoxide dismutase. Ascorbic acid increased AOA; however, EF and superoxide dismutase did not change AOA compared with sham exposure in stressed rats. No influence on the lipid peroxide level and AOA in unstressed rats was observed with EF exposure alone. Although the administration of AAPH decreased AOA, this decrease did not change when EF was added. These data indicate that the ELF EF used in this study influenced the lipid peroxide level in an oxidatively stressed rat.     

Isolation of protoplast from aerial mycelia and fruit-body formation from regenerated mycelia in Pleurotus cornucopiae

We have developed a simple and efficient method for protoplast isolation from Pleurotus cornucopiae. Protoplasts were isolated from aerial mycelia cultured on potato dextrose agar medium without time-consuming propagation in liquid culture. Protoplast yield was significantly increased by means of a decompressing pretreatment of mycelia in enzyme solution and a subsequent enzyme reaction with vibrational mixing. The isolated protoplasts regenerated mycelia and these mycelia formed fruit-bodies without any morphological abnormalities.     

Smad3 and Snail show circadian expression in human gingival fibroblasts, human mesenchymal stem cell, and in mouse liver

Smads are intracellular signaling mediators. Complexes of Smad2 and Smad3 with Smad4 transmit transforming growth factor-beta (TGF-β) receptor-induced signaling. Snail plays important roles in mesoderm formation, gastrulation, neural crest development, and epithelial mesenchymal transition. However, it remains unknown whether Smad3 and Snail expression is circadian rhythm-dependent. Here, we showed for the first time that Smad3 and Snail show circadian expression in human gingival fibroblasts (HGF-1) and human mesenchymal stem cells (MSC) after serum shock. They also showed circadian expression in the mouse liver. We confirmed that BMAL1/2, DEC1/2, VEGF, and PER1/2/3 also show circadian expression in both HGF-1 and MSC. The mRNA peaks and phases in circadian expression of these genes differed between HGF-1 and MSC. In a luciferase assay, Smad3 promoter activity was upregulated by CLOCK/BMAL1. These findings suggest that Smad3 and Snail have circadian rhythm in vitro and vivo, and that circadian expression of Smad3 depends on CLOCK/BMAL1.